Tim protein-bound carrier, methods for obtaining, removing and detecting extracellular membrane vesicles and viruses using said carrier, and kit including said carrier

ABSTRACT

The invention provides a carrier and a method for obtaining, removing, or detecting extracellular membrane vesicle or virus present in a sample. In particular, the invention provides (a) a carrier (a Tim carrier) on which a protein (a Tim protein), selected from a T-cell immunoglobulin and mucin domain-containing molecule-4 (a Tim-4) protein, a Tim-3 protein, and a Tim-1 protein, is bound; (b) a method for obtaining the extracellular membrane vesicle or the virus in the sample; (c) a method for removing the extracellular membrane vesicle or the virus in the sample; (d) a method for detecting the extracellular membrane vesicle or the virus in the sample; (e) a kit for capturing the extracellular membrane vesicle or the virus, comprising the Tim carrier; and f) a kit for capturing the extracellular membrane vesicle or the virus, comprising a reagent containing the Tim protein and a reagent containing the carrier.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is the U.S. national phase of InternationalPatent Application No. PCT/JP2015/083505, filed Nov. 27, 2015, whichclaims the benefit of Japanese Patent Application No. 2014-246876, filedon Dec. 5, 2014, which are incorporated by reference in their entiretiesherein.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 56,202 bytes ASCII (Text) file named“728241ReplacementSequenceListing.txt,” created Sep. 12, 2017.

TECHNICAL FIELD

The present invention relates to a Tim protein-binding carrier, a methodfor obtaining, removing and detecting extracellular membrane vesiclesand viruses using the carrier, as well as a kit comprising the carrier.

BACKGROUND ART

It has been known that the extracellular membrane vesicles containproteins or nucleic acids, such as microRNAs, and the like, inside ofthe particles, and are responsible for transferring substances betweencells. The extracellular membrane vesicles are secreted also into bodyfluids, such as blood, and the like, and proteins, microRNAs, and thelike, in the extracellular membrane vesicles have been noticed asdiagnostic markers for diseases. In addition, application of theextracellular membrane vesicles as a delivery tool of nucleic acidmedicines has also been noticed.

In addition, there are many enveloped viruses covered with amembrane-like envelope on the surface of the viruses. Many envelopedviruses, such as influenza virus, human immunodeficiency virus, and thelike, have been noticed as research subjects, since they cause diseases.In addition, the enveloped viruses are detoxified and utilized forvaccines, vectors, and the like, and they have been applied inprevention and treatment of diseases.

In this way, a process for obtaining the extracellular membrane vesiclesor the enveloped viruses in high purity is indispensable, in theutilization of the extracellular membrane vesicles for diagnosis andmedicines, in the utilization of the enveloped viruses as the vaccinesand the vectors, and in carrying out basic research, or the like, offunctional analysis, or the like, of the extracellular membrane vesiclesor the enveloped viruses, and thus a means for obtaining theextracellular membrane vesicles or the enveloped viruses easily andconveniently, as well as in high purity has long been awaited. Inaddition, development of a method for efficiently removing theextracellular membrane vesicles or the enveloped viruses from materialsderived from body fluid to be used has been expected, to preventcontamination of the extracellular membrane vesicles or the envelopedviruses derived from living organisms. Furthermore, a method fordetecting the obtained extracellular membrane vesicles or the envelopedviruses, or the extracellular membrane vesicles or the enveloped virusesin a specimen in high sensitivity has also long been awaited.

As a method for obtaining the extracellular membrane vesicles, there hasbeen known, as the most common method, a method in which a sample issubjected to ultracentrifugal separation treatment to obtain theextracellular membrane vesicles as a precipitate fraction (NON-PATENTLITERATURE 1). However, with this method, it is difficult to obtain thehighly pure extracellular membrane vesicles, because protein complexesor aggregates, lipoproteins, such as HDL, and the like, contained in asample are also co-precipitated, other than the extracellular membranevesicles. Although it is possible to separate the protein complexes orthe aggregates by density fractionation of the precipitate fractionobtained by the ultracentrifugal separation treatment by a sucrosedensity gradient method, it is difficult to separate them from the HDLhaving the same density. In addition, it is difficult to processmultiple samples at the same time, since this method requiresultracentrifugal separation treatment. In addition, expensive equipmentis required for ultracentrifugal separation treatment.

In addition, there is also a method for obtaining the extracellularmembrane vesicles, as a precipitated fraction, by centrifugal separationtreatment by the addition of a commercial reagent, represented byExoQuick (produced by System Biosciences Co., Ltd.) or Total ExosomeIsolation Reagent (produced by Thermo Fisher Scientific Co., Ltd.)(NON-PATENT LITERATURE 1), however, there is a problem that purity ofthe resulting extracellular membrane vesicles is further lower ascompared with a method by the ultra-centrifugal separation treatment.

In addition to these conventional methods, there are methods forobtaining the extracellular membrane vesicles by affinity of a surfaceantigenic protein and an antibody, using the antibody against thesurface antigenic protein of the extracellular membrane vesicles (ananti-CD63 antibody immobilization method, Exosome-Human CD63Isolation/Detection (produced by Thermo Fisher Scientific, Inc.), andthe like.) (NON-PATENT LITERATURE 1). These methods have such problemsthat only the extracellular membrane vesicles having the surfaceantigenic protein for the antibody can be obtained; yield of theextracellular membrane vesicles is small; it is difficult to obtain theextracellular membrane vesicles in an intact state (that is, whilemaintaining an original state of function of the extracellular membranevesicles) because it is necessary to use a surfactant, an acidic buffer,or the like, in order to elute the extracellular membrane vesicles fromthe antibody; and the like, although the high purity extracellularmembrane vesicles can be obtained.

As a method for obtaining the viruses, there has been known, as the mostgeneral method, a method for subjecting a sample to ultracentrifugalseparation treatment to obtain the viruses, as the precipitate fraction(NON-PATENT LITERATURE 1). However, in this method, it is difficult toobtain the high purity viruses, because the protein complexes and theaggregates, the lipoproteins, such as HDL, and the like, contained inthe sample are co-precipitated other than the viruses. Although it ispossible to separate the protein complexes or the aggregates by densityfractionation of the precipitate fraction obtained by theultracentrifugal separation treatment, by a sucrose density gradientmethod, it is difficult to separate them from the aggregates having thesame density. In addition, there is also a problem that activity of theviruses is lowered by the ultracentrifugal separation treatment(NON-PATENT LITERATURE 2). In addition, this method is difficult toprocess multiple specimens at the same time, because it requires theultracentrifugal separation treatment. In addition, an expensive machineis required for the ultracentrifugal separation treatment.

Other than these methods, there has been known a method for purifyingthe viruses by ion-exchange chromatography, (NON-PATENT LITERATURE 3).However, this method requires setting optimum condition for each of theviruses. In addition, condition setting may be difficult, in some cases,and thus it is difficult to apply it to purification of all viruses.

As a method for removing the extracellular membrane vesicles, there hasbeen known, as the most general method, a method for subjecting thesample to the ultra-centrifugal separation treatment to precipitationfractionate the extracellular membrane vesicles to obtain thesupernatant fraction as a removal sample. However, this method hasdifficulty in complete removal of the extracellular membrane vesicles,thus resulting in remaining of the extracellular membrane vesicles notcompletely removed, in the sample.

Furthermore, there is also a method for precipitation fractioning theextracellular membrane vesicles, by the centrifugal separation treatmentand the addition of a commercial reagent, represented by ExoQuick(produced by System Biosciences Co., Ltd.) or Total Exosome IsolationReagent (produced by Thermo Fisher Scientific Co., Ltd.) to obtain thesupernatant fraction as the removal sample. However, in also thismethod, there was difficulty to completely remove the extracellularmembrane vesicles, due to remaining of the extracellular membranevesicles not completely removed, in the sample. In addition, there wasthe case of inducing a problem on bioactivity, or the like, of theextracellular membrane vesicles, by the reagent, because ofcontamination of the reagent added into the sample.

There has been known the same method also as a method for removing theviruses and there was the same problem as in the method for removing theextracellular membrane vesicles.

As a method for detecting the extracellular membrane vesicles, there hasbeen known, as a general method, a sandwich ELISA using the antibodyagainst the surface antigen of the extracellular membrane vesicles.However, the minimum detection sensitivity of the sandwich ELISA systemusing the antibody that detects an ordinary color development signal hasbeen reported to be about 3 μg in purified exosomes, and sufficientsensitivity for measuring a body fluid sample, such as serum, has notbeen obtained (NON-PATENT LITERATURE 4). The same method has been known,also as the method for detecting the viruses, and sufficient sensitivityfor detection of the viruses has also not been obtained.

In addition, as the other method for detecting the extracellularmembrane vesicles, a flow cytometry method has been known. However, inthe usual method for capturing the extracellular membrane vesicles usinga carrier to which the antibody against the surface antigen of theextracellular membrane vesicles was immobilized, sufficient sensitivityhas not been obtained; and thus direct detection from the sample, suchas culture supernatant or body fluid, has been difficult, and thedetection from the sample after concentrating or purifying theextracellular membrane vesicles has been required.

CITATION LIST Non-Patent Literature

-   [NON-PATENT LITERATURE 1] Kenneth W. Witwer et al. Journal of    Extracellular Vesicles, 2013 May 27; 2. doi: 10.3402-   [NON-PATENT LITERATURE 2] G. Y. Chen et al. Biotechnol. Prog. 25    (2009), 1669-   [NON-PATENT LITERATURE 3] Petra Gerster et al. Journal of    Chromatography A, 1290 (2013), 36-45-   [NON-PATENT LITERATURE 4] Mariantonia Logozzi et al. PLoS ONE 4    (2009), 5219

SUMMARY OF INVENTION Technical Problem

As described above, the conventional methods for obtaining theextracellular membrane vesicles or the viruses have difficulty inobtaining the highly pure extracellular membrane vesicles or the virusesin an intact state, simply and conveniently, as well as efficiently. Inaddition, with the conventional methods for removing the extracellularmembrane vesicles or the viruses, it is difficult to efficiently removethe extracellular membrane vesicles or the viruses in the sample.Furthermore, the conventional methods for detecting the extracellularmembrane vesicles or the viruses have not achieved sufficientsensitivity.

Therefore, an object of the present invention is to obtain or remove theextracellular membrane vesicles or the viruses present in the sample inhigh purity, and in an intact state, simply and conveniently, as well asefficiently, and detect the extracellular membrane vesicles or theviruses in high sensitivity.

Solution to Problem

The present invention has been made to solve the problems, and has thefollowing constitution:

1. A carrier (a Tim carrier) on which a protein (a Tim protein) selectedfrom a T-cell immunoglobulin and mucin domain-containing molecule-4 (aTim-4) protein, a T-cell immunoglobulin and mucin domain-containingmolecule-3 (a Tim-3) protein, and a T-cell immunoglobulin and mucindomain-containing molecule-1 (a Tim-1) protein, is bound.2. A method for obtaining an extracellular membrane vesicle or a virusin a sample, comprising the following steps:(1) a step for forming a complex of the Tim protein bound to thecarrier, and the extracellular membrane vesicle or the virus in thesample, in the presence of a calcium ion (complex formation step),(2) a step for separating the complex from the sample (complexseparation step),(3) a step for separating the extracellular membrane vesicle or thevirus from the complex to obtain the extracellular membrane vesicle orthe virus (obtaining step).3. A method for removing an extracellular membrane vesicle or a virus ina sample, comprising the following steps:(1) a step for forming the complex of the Tim protein bound to thecarrier, and the extracellular membrane vesicle or the virus in thesample in the presence of the calcium ion (complex formation step),(2) a step for separating the complex and the sample (complex separationstep).4. A method for detecting an extracellular membrane vesicle or a virusin the sample, comprising the following steps:(1) a step for forming the complex of the Tim protein bound to thecarrier, and the extracellular membrane vesicle or the virus in thesample, in the presence of the calcium ion (complex formation step),(2) a step for detecting the complex (detection step).5. A kit for capturing an extracellular membrane vesicle or a viruscomprising the Tim carrier.6. A kit for capturing an extracellular membrane vesicle or a viruscomprising a reagent containing the Tim protein and a reagent containingthe carrier.

In view of the above circumstances, the present inventors havediscovered, as a result of intensive studies, that the extracellularmembrane vesicles or the viruses having phosphatidylserine on thesurface can be obtained in high purity, that the extracellular membranevesicles or the viruses can be obtained in an intact state, that theextracellular membrane vesicles or the viruses can be efficientlyremoved, and that they can be detected in high sensitivity, by using atleast one kind of a protein selected from the Tim-1 protein, the Tim-3protein and the Tim-4 protein, and have thus completed the presentinvention.

Phosphatidylserine, which is a phospholipid, is present (exposed) on thesurface of the extracellular membrane vesicles. As a protein havingbinding capability to phosphatidylserine (hereinafter it may beabbreviated as “the phosphatidylserine-binding protein” or “the PSproteins”, in some cases), there has been known, for example, Annexin V,MFG-E8, the Tim-1 (the T-cell immunoglobulin and mucin domain-containingmolecule-1, the T-cell immunoglobulin-mucin-domain 1), the Tim-1 (theT-cell immunoglobulin and mucin domain-containing molecule-1, the T-cellimmunoglobulin-mucin-domain-1) protein, the Tim-3 (the T-cellimmunoglobulin and mucin domain-containing molecule-3, the T-cellimmunoglobulin-mucin-domain-3) protein, the Tim-4 (the T-cellimmunoglobulin and mucin domain-containing molecule-4, the T-cellimmunoglobulin-mucin-domain-4) protein, or the like (The Journal ofBiochemistry 265, 4923-4928 (25 Mar. 1990), Nature 417, 182-187 (9 May2002), Nature 450, 435-439 (15 Nov. 2007).

However, it has been revealed that it is difficult to obtain, remove ordetect the extracellular membrane vesicles or the viruses, when the PSproteins other than the Tim-1 protein, the Tim-3 protein and the Tim-4protein are used, among these PS proteins.

Advantageous Effects of Invention

According to the present invention, the extracellular membrane vesiclesor the viruses present in the sample can be obtained or removed in highpurity, or in an intact state, simply and conveniently, as well asefficiently, and can be detected in high sensitivity.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an electropherogram for confirming whether the extracellularmembrane vesicles were obtained or not, by Western blotting, in Examples1 to 8.

FIG. 2 is an electropherogram for confirming whether the extracellularmembrane vesicles were obtained or not, by Western blotting, in Examples9 to 16.

FIG. 3 is an electropherogram for confirming whether the extracellularmembrane vesicles were obtained or not, by Western blotting, in Examples17 to 18.

FIG. 4-A is an electropherogram for confirming whether the extracellularmembrane vesicles were obtained or not, by Western blotting, in Examples19 to 20, and Comparative Examples 1 to 3, and FIG. 4-B is anelectropherogram for confirming whether the extracellular membranevesicles were obtained or not, by silver staining, in Examples 19 to 20,and Comparative Examples 1 to 3.

FIG. 5 is a drawing where the extracellular membrane vesicles, obtainedby the method of the present invention, were observed using an electronmicroscope, in Example 21.

FIG. 6 is an electropherogram for confirming whether the extracellularmembrane vesicles were obtained or not, by Western blotting, in Examples22 to 25.

FIG. 7 is an electropherogram for confirming whether the extracellularmembrane vesicles were obtained or not, by silver staining, in Examples26 to 27, and Comparative Examples 4 to 9.

FIG. 8 is an electropherogram for confirming whether the extracellularmembrane vesicles were obtained or not, by Western blotting, in Examples28 to 33.

FIG. 9 is an electropherogram for confirming whether the extracellularmembrane vesicles were obtained or not, by Western blotting, in Examples34 to 35, and Comparative Examples 10 to 13.

FIG. 10 is an electropherogram for confirming whether the extracellularmembrane vesicles were obtained or not, by Western blotting, in Examples36 to 38.

FIG. 11 is an electropherogram for confirming whether the extracellularmembrane vesicles were obtained or not, by Western blotting, in Examples39 to 40.

FIG. 12 is an electropherogram for confirming whether the extracellularmembrane vesicles were obtained (removed) or not, by Western blotting,in Examples 41 to 47.

FIG. 13 is an electropherogram for confirming whether the extracellularmembrane vesicles were obtained (removed) or not, by Western blotting,in Examples 48 to 55.

FIG. 14 is an electropherogram for confirming whether the extracellularmembrane vesicles were obtained or not, by Western blotting, in Examples56 to 67, and Comparative Examples 14 to 15.

FIG. 15 is an electropherogram for confirming whether the extracellularmembrane vesicles were obtained or not, by Western blotting, in Examples68 to 79.

FIG. 16 is an electropherogram for confirming whether the extracellularmembrane vesicles were obtained or not, by Western blotting, in Examples80 to 83, and Comparative Examples 16 to 19.

FIG. 17 is an electropherogram for confirming whether the extracellularmembrane vesicles were obtained or not, by Western blotting, in Examples84 to 95, and Comparative Examples 20 to 21.

FIG. 18 is an electropherogram for confirming whether the extracellularmembrane vesicles were obtained or not, by Western blotting, in Examples96 to 99, and Comparative Examples 22 to 25.

FIG. 19 is detection result of the extracellular membrane vesicles by anELISA method, in Examples 100 to 105, and Comparative Examples 26 to 33.

FIG. 20 is detection result of the extracellular membrane vesicles bythe ELISA method, in Examples 106 to 109.

FIG. 21 is detection result of the extracellular membrane vesicles bythe ELISA method, in Examples 110 to 115, and Comparative Examples 34 to39.

FIG. 22 is an electropherogram for confirming whether the extracellularmembrane vesicles were obtained or not, by Western blotting, in Examples116 to 119.

FIG. 23 is detection result of the extracellular membrane vesicles by aflow cytometry method, in Examples 120 to 121, and Comparative Examples40 to 41.

FIG. 24 is an electropherogram for confirming whether the extracellularmembrane vesicles were obtained or not, by Western blotting, in Examples122 to 123, and Comparative Examples 42 to 43

FIG. 25 is an electropherogram for confirming whether the extracellularmembrane vesicles were obtained or not, by Western blotting, in Examples124 to 125, and Comparative Examples 44 to 45.

DESCRIPTION OF EMBODIMENTS

<1. Extracellular Membrane Vesicle Pertaining to the Present Invention>

The extracellular membrane vesicle pertaining to the present inventionis the small-sized membrane vesicle secreted from in vivo cells orcultured cells, composed of a lipid bilayer membrane, and havingphosphatidylserine on the membrane surface thereof. Diameter of thevesicle is usually 20 nm to 1000 nm, preferably 50 nm to 500 nm, andmore preferably 50 nm to 200 nm.

The extracellular membrane vesicle pertaining to the present inventioninclude those classified variously depending on origin of its generationand size of the small-sized membrane vesicle, and the like, as describedin Nature Reviews Immunology 9, 581-593 (August, 2009), “Obesity Study”Vol. 13, No. 2, 2007, topics by Naoto Aoki et al. Specifically, thereare included Exosomes, micro-vesicles, Ectosomes, Membrane particles,Exosome-like vesicles, Apoptotic vesicles, Adiposome, and the like.

Exosome is the small-sized membrane vesicle derived from late endosomecomposed of the lipid bilayer membrane, and having phosphatidylserine onthe membrane surface thereof. Diameter of the vesicle is usually 50 nmto 200 nm, preferably 50 nm to 150 nm, and more preferably 50 nm to 100nm. Exosome is known to contain proteins, such as tetraspanins, CD 63,CD 9, and the like, Alix, TSG 101, Lamp-1, Flotillin, and the like.

The micro-vesicle is the small-sized membrane vesicle derived from acell membrane (plasma membrane), composed of the lipid bilayer membrane,and having phosphatidylserine on the membrane surface thereof. Themicro-vesicle is usually 100 nm to 1000 nm, preferably 100 nm to 800 nm,and more preferably 100 nm to 500 nm. The micro-vesicle is known tocontain proteins, such as integrin, selectin, CD40 ligands, and thelike.

Ectosome is the small-sized membrane vesicle derived from the cellmembrane (plasma membrane), composed of the lipid bilayer membrane, andhaving phosphatidylserine on the membrane surface thereof. Ectosome isusually 50 nm to 200 nm, preferably 50 nm to 150 nm, and more preferably50 nm to 100 nm. Ectosome is known to contain CR1, a proteolytic enzyme,but does not contain CD63.

The membrane particle is the small-sized membrane vesicle derived fromthe cell membrane (plasma membrane), composed of the lipid bilayermembrane, and having phosphatidylserine on the membrane surface thereof.The membrane particle is usually 50 nm to 80 nm. It is known that themembrane particle contains CD133 but does not contain CD63.

The exosome-like vesicle is the small-sized membrane vesicle derivedfrom the early endosome, composed of the lipid bilayer, and havingphosphatidylserine on the membrane surface thereof. The exosome-likevesicle is usually 20 nm to 50 nm. The exosome-like vesicle is known tocontain TNFRI.

The apoptotic vesicle is the small-sized membrane vesicle derived fromthe apoptotic cell, composed of the lipid bilayer, and havingphosphatidylserine on the membrane surface thereof. The apoptoticvesicle is usually 50 nm to 500 nm, preferably 50 nm to 300 nm, and morepreferably 50 nm to 200 nm. The apoptotic vesicle is known to containhistones.

Adiposome is the small-sized membrane vesicle derived from theadipocyte, composed of the lipid bilayer membrane, and havingphosphatidylserine on the membrane surface thereof. The adiposome isusually 100 nm to 1000 nm, preferably 100 nm to 800 nm, and morepreferably 100 nm to 500 nm. The adiposome is known to contain MFG-E8(milk fat globule-EGF factor 8).

<2. Virus Pertaining to the Present Invention>

The virus pertaining to the present invention has an envelope composedof the lipid bilayer membrane derived from a cell membrane, a nuclearmembrane, a Golgi apparatus, an endoplasmic reticulum, or the like, of ahost cell as a capsid (outer shell) of the virus, and hasphosphatidylserine on the surface of the envelope (hereinafter referredto as “the enveloped virus”). Diameter of the virus pertaining to thepresent invention is usually 20 nm to 320 nm. The enveloped viruspertaining to the present invention includes the virus having anenvelope, belonging to a family described in the Biochemical Dictionary2nd edition, Tokyo Kagaku Dojin, 1990, 1503p-1505p. Specifically, thereare included Poxviridae, Baculoviridae, Rhabdoviridae, Bunyaviridae,Togaviridae, Herpesviridae, Paramyxoviridae, Orthomyxoviridae,Retroviridae, Arenaviridae, Coronaviridae, and the like.

<3. Tim Protein Pertaining to the Present Invention>

The Tim protein pertaining to the present invention is at least one kindof the Tim protein selected from the T-cell immunoglobulin and mucindomain-containing molecule-1 (the Tim-1) protein pertaining to thepresent invention (hereinafter it may be abbreviated as “the Tim-1protein pertaining to the present invention”, in some cases), the T-cellimmunoglobulin and mucin domain-containing molecule-3 (the Tim-3)protein pertaining to the present invention (hereinafter it may beabbreviated as “the Tim-3 protein pertaining to the present invention”,in some cases), and the T-cell immunoglobulin and mucindomain-containing molecule-4 (the Tim-4) protein pertaining to thepresent invention (hereinafter it may be abbreviated as “the Tim-4protein pertaining to the present invention”, in some cases).

The Tim-4 (the T-cell immunoglobulin and mucin domain-containingmolecule-4) protein pertaining to the present invention may be anyprotein capable of binding to the extracellular membrane vesicles or theviruses pertaining to the present invention, and the Tim-4 proteinderived from an animal is preferable. Among these, the Tim-4 protein ofhuman or a mouse is preferable (hereinafter, the Tim-4 protein of humanmay be abbreviated as “the human-derived Tim-4 protein”, and the Tim-4protein of mouse may be abbreviated as “the mouse-derived Tim-4protein”, in some cases).

More specifically, it may be the one, as long as having at least anamino acid sequence of the binding domain (IgV domain) forphosphatidylserine, and it may be the one having a full-length aminoacid sequence of the Tim-4 protein, or a part of the amino acid sequenceof the Tim-4 protein.

The amino acid sequence of the binding domain (IgV domain) forphosphatidylserine includes, for example, SEQ ID NO: 1 (N-terminal 22 to135 amino acid region (RefSeq NP_848874.3) of the mouse-derived Tim-4protein), SEQ ID NO: 2 (N-terminal 25 to 137 amino acid region (RefSeqNP_612388.2) of the human-derived Tim-4 protein), or the like.

The full-length amino acid sequence of the Tim-4 protein includes SEQ IDNO: 3 (full-length sequence of the mouse-derived Tim-4 protein1 to 343amino acid region (RefSeq NP_848874.3)), SEQ ID NO: 4 (full lengthsequence of the human-derived Tim-4 protein 1 to 378 amino acid region(RefSeq NP_612388.2)), or the like.

A part of the Tim-4 protein includes those having the amino acidsequence of the binding domain (IgV domain) for phosphatidylserine andthe mucin domain, such as SEQ ID NO: 5 (N-terminal 22 to 273 amino acidregion of the mouse-derived Tim-4 protein (RefSeq NP_848874.3)), SEQ IDNO: 6 (N-terminal 22 to 279 amino acid region of the mouse-derived Tim-4protein (RefSeq NP_848874.3)), SEQ ID NO: 7 (N-terminal 25 to 315 aminoacid region of the human-derived Tim-4 protein (RefSeq NP_612388.2)), orthe like. These sequences may have a signal sequence, as needed.

The Tim-1 (the T-cell immunoglobulin and mucin domain-containingmolecule-1) protein pertaining to the present invention may be anyprotein, as long as capable of binding to the extracellular membranevesicles or the viruses pertaining to the present invention, and theTim-1 protein derived from an animal is preferable. Among these, theTim-1 protein of human or mouse is preferable (hereinafter, sometimesthe Tim-1 protein of human is abbreviated as “the human-derived Tim-1protein” and the Tim-1 protein of mouse is abbreviated as “themouse-derived Tim-1 protein”).

More specifically, it may be the one, as long as having at least theamino acid sequence of the binding domain (IgV domain) forphosphatidylserine, and it may be the one having a full-length aminoacid sequence of the Tim-1 protein, or a part of the Tim-1 protein.

The amino acid sequence of the binding domain (IgV domain) forphosphatidylserine include, for example, SEQ ID NO: 8 (N-terminal 22 to131 amino acid region (RefSeq NP_001160104.1) of the mouse-derived Tim-1protein, SEQ ID NO: 9 (N-terminal 21 to 130 amino acid region (RefSeqNP_036338.2), or the like.

The full-length amino acid sequence of the Tim-1 protein includes SEQ IDNO: 10 (full-length sequence 1 to 282 amino acid region of themouse-derived Tim-1 protein (RefSeq NP_001160104.1)), SEQ ID NO: 11(full length sequence of the human-derived Tim-1 protein 1 to 364 aminoacid region (RefSeq NP_036338.2)), or the like.

A part of the Tim-1 protein includes those having the amino acidsequence of the binding domain (IgV domain) for phosphatidylserine andthe mucin domain, such as SEQ ID NO: 12 (N-terminal 22 to 212 amino acidregion of the mouse-derived Tim-1 protein (RefSeq NP_001160104.1)), SEQID NO: 13 (N-terminal 21 to 295 amino acid region of the human-derivedTim-1 protein (RefSeq NP_612388.2), or the like. These sequences mayhave the signal sequence, as needed.

The Tim-3 (the T-cell immunoglobulin and mucin domain-containingmolecule-3) protein pertaining to the present invention may be anyprotein, as long as capable of binding to the extracellular membranevesicles or the viruses pertaining to the present invention, and theTim-3 protein derived from an animal is preferable. Among these, theTim-3 protein of human or mouse is preferable (hereinafter, the Tim-3protein of human is abbreviated as “the human-derived Tim-3 protein”,and the Tim-3 protein of mouse is abbreviated as “the mouse-derivedTim-3 protein”, in some cases).

More specifically, it may be the one, as long as having at least theamino acid sequence of the binding domain (IgV domain) forphosphatidylserine, and it may be the one having a full-length aminoacid sequence of the Tim-3 protein, or a part of the Tim-3 protein.

The amino acid sequence of the binding domain (IgV domain) forphosphatidylserine include, for example, SEQ ID NO: 14 (N-terminal 22 to134 amino acid region (RefSeq NP_599011.2) of the mouse-derived Tim-3protein), SEQ ID NO: 15 (N-terminal 22 to 135 amino acid region (RefSeqNP_116171.3) of the human-derived Tim-3 protein), or the like.

The full-length amino acid sequence of the Tim-3 protein includes SEQ IDNO: 16 (full-length sequence 1 to 281 amino acid region of themouse-derived Tim-3 protein (RefSeq NP_599011.2)), SEQ ID NO: 17 (fulllength sequence of the human-derived Tim-3 protein 1 to 301 amino acidregion (RefSeq NP_116171.3)), or the like.

A part of the Tim-3 protein includes those having the amino acidsequence of the binding domain (IgV domain) for phosphatidylserine andthe mucin domain, such as SEQ ID NO: 18 (N-terminal 22 to 189 amino acidregion of the mouse-derived Tim-3 protein (RefSeq NP_599011.2)), SEQ IDNO: 19 (N-terminal 22 to 200 amino acid region of the human-derivedTim-3 protein (RefSeq NP_116171.3), or the like. These sequences mayhave the signal sequence, as needed.

In addition, the Tim protein pertaining to the present invention may bea mutant of the above amino acid sequences, in which one or more aminoacids are deleted, substituted, inserted and/or added, as long as it iscapable of binding to the extracellular membrane vesicles or the virusespertaining to the present invention.

The Tim protein pertaining to the present invention may be the one, aslong as having properties as described above, and may be the oneextracted from cells (for example, immune cells, such as macrophages) ortissues of living organisms having the Tim protein, such as animalsincluding the mouse and the human, or plants, and the like, and the oneprepared by genetic recombination technology based on this, or the like,and any of them can be used.

In using the Tim protein pertaining to the present invention prepared bygenetic recombination technology, those having one or more affinity tagsare preferable, from the viewpoint of simplicity of purification.

The affinity tags may be any tags, as long as being used in preparationof a protein by genetic recombination technology, and include theaffinity tags, for example, Fc tag, FLAG tag, His tag, GST tag, MBP tag,HA tag, Myc tag, Strep (II) tag, PA tag, and the like.

The affinity tags are fused to the C-terminal side of the Tim proteinpertaining to the present invention.

Therefore, the Tim protein pertaining to the present invention includesnot only a protein consisting only of the amino acid sequence of the Timprotein (full length or partial sequence) but also a protein having theamino acid sequence (full length or partial sequence) of the Tim proteinand the amino acid sequence of the affinity tag, as described above.

In addition, the affinity tag and the Tim protein may be bound directlyto each other, or may be bound via a spacer as described in “ProteinExpression using a cell-free protein synthesis reagent kit, Transdirectinsect cell, derived from insect cultured cells”, Toru Ezure, TakashiSuzuki, Masaaki Ito, Masamitu Shikata (Shimadzu Seisakusho, AnalyticalMeasurement Division), publication date 2008 Jun. 9: Protein ScienceFoundation Archive, 1, e005 (2008)”, or the like. Therefore, the Timprotein pertaining to the present invention also includes a proteinhaving the amino acid sequence (full length or partial sequence) of theTim protein, and the amino acid sequence of the affinity tag asdescribed above, and the amino acid sequence of the spacer.

<4. Preparation Method for Tim Protein Pertaining to the PresentInvention>

The Tim protein pertaining to the present invention can be produced by ageneral chemical production method, in accordance with the amino acidsequence thereof. The Tim protein pertaining to the present inventioncan be obtained by a usual chemical production method (chemicalsynthesis method), for example, a fluorenylmethyloxycarbonyl method(Fmoc method), a t-butyloxycarbonyl method (t-Boc method), or the like.In addition, it can be chemically synthesized also, using a commerciallyavailable peptide synthesizer.

Furthermore, the Tim protein pertaining to the present invention canalso be obtained by a well-known method using the genetic recombinationtechnique, such that a nucleic acid molecule encoding the Tim proteinpertaining to the present invention is incorporated into an expressionvector, such as an appropriate plasmid or phage, the host cell istransformed (transduced) using the recombinant expression vector, andthe resulting host cell is proliferated to secrete the Tim proteininside of the cell or outside of the cell.

Explanation will be given below on the preparation method for the Timprotein pertaining to the present invention, for the case of preparationby the genetic recombination technology.

<Expression Vector Pertaining to the Present Invention>

The expression vector for expressing the Tim protein pertaining to thepresent invention (hereinafter, it is referred to as the expressionvector pertaining to the present invention) may be any one, as long asit contains the nucleic acid sequence encoding the Tim proteinpertaining to the present invention (hereinafter, it may be abbreviatedas “Tim coding sequence pertaining to the present invention”, in somecases).

Among the Tim coding sequences pertaining to the present invention, thenucleic acid sequence encoding the Tim-4 protein includes, for example,SEQ ID NO: 20 (the nucleotide sequence of cDNA encoding the full-lengthsequence 1 to 343 amino acid region of the mouse-derived Tim-4 protein(RefSeq No. NM_178759.4), and containing a stop codon (tga) at theterminal 3 nucleotides), SEQ ID NO: 21 (nucleotide sequence of cDNAencoding the full-length sequence 1 to 378 amino acid region of thehuman-derived Tim-4 protein (RefSeq No. NM_138379.2), and containing thestop codon (taa) at the terminal 3 nucleotides), or the like.

The nucleic acid sequence encoding the Tim-1 protein pertaining to thepresent invention includes, for example, SEQ ID NO: 22 (the nucleotidesequence of cDNA encoding the full-length sequence 1 to 282 amino acidregion of the mouse-derived Tim-1 protein (RefSeq No. NM_001166632.1),containing the stop codon (tga) at the terminal 3 nucleotides), SEQ IDNO: 23 (the nucleotide sequence of cDNA encoding the full-lengthsequence 1 to 364 amino acid region of the human-derived Tim-1 protein(RefSeq No. NM_012206.3), containing the stop codon (taa) at theterminal 3 nucleotides), or the like.

The nucleic acid sequence encoding the Tim-3 protein pertaining to thepresent invention includes, for example, SEQ ID NO: 24 (the nucleotidesequence of cDNA encoding the full-length sequence 1 to 281 amino acidregion of the mouse-derived Tim-3 protein (RefSeq No. NM_134250.2),containing the stop codon (tga) at the terminal 3 nucleotides), SEQ IDNO: 25 (the nucleotide sequence of the cDNA encoding the full-lengthsequence 1 to 301 the amino acid region of the human derived Tim-3protein (RefSeq No. NM_032782.4), containing the stop codon (tag) at theterminal 3 nucleotides), or the like.

For the expression vector pertaining to the present invention, theTim-coding sequence pertaining to the present invention may be geneintroduced into a commercially available vector, according to aconventional cloning method. The expression vector pertaining to thepresent invention includes, for example, an expression vector, such as acommercially available pCAG-Neo vector (produced by Wako Pure ChemicalIndustries, Ltd.), or the like, into which is incorporated SEQ ID NO: 26(cDNA encoding the N-terminal 1 to 273 amino acid region of themouse-derived Tim-4 protein, containing the stop codon (tga) at theterminal 3 nucleotides), SEQ ID NO: 27 (cDNA encoding the N-terminal 1to 279 amino acid region of the mouse-derived Tim-4 protein, containingthe stop codon (tga) at the terminal 3 nucleotides), SEQ ID NO: 28 (cDNAencoding the N-terminal 1 to 315 amino acid region of the human-derivedTim-4 protein, containing the stop codon (tga) at the terminal 3nucleotides), SEQ ID NO: 29 (cDNA encoding the N terminal 1 to 212 aminoacid region of the mouse-derived Tim-1 protein, containing the stopcodon (tga) at the terminal 3 nucleotides), SEQ ID NO: 30 (cDNA encodingthe N-terminal 1 to 295 amino acid region of the human-derived Tim-1protein, containing the stop codon (tga) at the terminal 3 nucleotides),SEQ ID NO: 31 (cDNA encoding N-terminal 1 to 189 amino acid region ofthe mouse-derived Tim-3 protein, containing the stop codon (tga) at theterminal 3 nucleotides), or SEQ ID NO: 32 (cDNA encoding N-terminal 1 to200 amino acid region of the human-derived Tim-3 protein, containing thestop codon (tga) at the terminal 3 nucleotides), in accordance with theconventional cloning technique. As the vector into which the Tim codingsequence is gene introduced, any vector may be used, as long as it hasfunction of expressing and producing the Tim protein pertaining to thepresent invention in the host cell, and it is convenient to usecommercially available vectors. The commercially available vectors usedfor this purpose include pCAG-Neo vector, pcDNA vector, and the like,when the host is an animal cell.

<Host>

As the host, anyone can be used, as long as it is capable of expressingthe Tim protein pertaining to the present invention, and includesEscherichia coli, insect cells, mammalian cells, plant cells, yeastcells, or the like, and the mammalian cells are preferable. Themammalian cells include, for example, HEK 293T cell, COS-7 cell, CHO-K1cell, CHO-S cell, and the like.

<Introduction of Gene into Host>

The expression vector pertaining to the present invention is geneintroduced into the host, according to a usual method of the techniqueof gene introducing the vector into the host, described in “Proteinexpression protocol that can be selected depending on each purpose,Chapter 3, Protein expression protocol, ISBN 978-4-7581-0175-2, YodoshaCo., Ltd.”, or the like.

<Culture of Host>

The host after carrying out gene introduction is cultured, according toa usual method for culturing the host. Culture condition variesdepending on the host into which the gene has been introduced, but maybe in accordance with a usual method for each host. For example, when itis an animal cell, the cell may be cultured for 1 day to 10 days, andpreferably 3 days to 4 days, under conditions of usually 5 to 10% CO₂,preferably 5 to 8% CO₂, usually at 36° C. to 38° C., preferably at 36.5°C. to 37.5° C. It should be noted that, the Tim protein pertaining tothe present invention is expressed and secreted in the culturesupernatant, since it does not contain a transmembrane domain and anintracellular domain.

<Purification of Tim Protein Pertaining to the Present Invention>

A culture supernatant filtrate may be obtained by subsequentlysubjecting a culture solution of the resulting gene-introduced host tocentrifugal separation treatment (usually at 200 to 400×G for 3 minutesto 10 minutes, and preferably at 300×G for 3 minutes to 6 minutes) torecover a culture supernatant, and subjecting, as needed, the recoveredculture supernatant to (i) centrifugal separation treatment usually at1000 to 2000×G for 20 minutes to 60 minutes, and preferably at 1200×Gfor 20 minutes to 40 minutes, and/or to (ii) filtration treatment toseparate impurities.

Further, a concentrated culture supernatant filtrate may be obtained byconcentration of the resulting culture supernatant filtrate usually 5times to 20 times, and preferably 8 times to 12 times, according to ausual method, such as ultrafiltration, or the like, as needed.

Next, when the Tim protein pertaining to the present invention has theaffinity tag, the Tim protein (a fusion protein of the Tim protein andthe affinity tag) pertaining to the present invention may be purifiedfrom the resulting culture supernatant, from the resulting culturesupernatant filtrate, or from the resulting concentrated solution of theculture supernatant filtrate, according to a usual method for proteinpurification utilizing the affinity tag (for example, a method for usinga carrier on which a substance having affinity to the affinity tag isimmobilized) for each tag, described in “Protein expression protocolthat can be selected depending on each purpose, Chapter 3 Proteinexpression protocol, Section 6, Purification of protein purification bya tag, ISBN 978-4-7581-0175-2, Yodosha Co., Ltd.”, or the like.

When the Tim protein pertaining to the present invention does not havethe affinity tag, the Tim protein pertaining to the present inventionmay be purified by various kinds of chromatography, from the resultingculture supernatant, from the resulting culture supernatant filtrate, orfrom the resulting concentrated solution of the culture supernatantfiltrate, according to a usual method of protein purification, describedin “Protein expression protocol that can be selected depending on eachpurpose, Chapter 3, Protein expression protocol, Section 6, Purificationof protein Purification by chromatography, ISBN 978-4-7581-0175-2,Yodosha Co., Ltd.”, or the like.

Purification may be carried out by appropriately combining the abovepurification methods.

<Specific Preparation Method for Tim Protein Pertaining to the PresentInvention>

As a specific preparation method for the Tim protein pertaining to thepresent invention, for example, when a Fc tag is used as the affinitytag, the following method is included. First, according to a usualmethod, the Tim coding sequence pertaining to the present invention isincorporated into a pEF-Fc vector or a commercially available vector toconstruct the expression vector pertaining to the present invention.Subsequently, the expression vector pertaining to the present inventionis gene introduced into the host cell, according to a usual method toculture for 1 day to 10 days, and preferably 3 days to 4 days, underconditions of usually 5 to 10% CO₂, preferably 5 to 8% CO₂, at 36° C. to38° C., preferably 36.5° C. to 37.5° C. The culture supernatant filtrateis obtained by subsequently subjecting the resulting culture solution ofthe gene-introduced host to a centrifugal separation treatment (usuallyat 200 to 400×G for 3 minutes to 10 minutes, and preferably at 300×G for3 minutes to 6 minutes) to recover the culture supernatant, and bysubjecting, as needed, the recovered culture supernatant to (i)centrifugal separation treatment usually at 1000 to 2000×G for 20minutes to 60 minutes, and preferably at 1200×G for 20 minutes to 40minutes, and/or to (ii) filtration treatment to separate impurities.Further, the concentrated solution of the culture supernatant filtrateis obtained by concentration, as needed, of the resulting culturesupernatant filtrate usually 5 times to 20 times, and preferably 8 timesto 12 times, according to a usual method, such as ultrafiltration, orthe like. After that, when the Tim protein pertaining to the presentinvention has the affinity tag, the Tim protein pertaining to thepresent invention can be obtained by purifying the Tim proteinpertaining to the present invention from the resulting culturesupernatant, the resulting culture supernatant filtrate, or theresulting concentrated solution of the culture supernatant filtrate, inaccordance with a usual method for purification utilizing affinity ofrespective affinity tags.

<5. Tim Carrier of the Present Invention>

A carrier on which the Tim protein of the present invention is bound(hereafter it may be abbreviated as “the Tim carrier of the presentinvention”, in some cases) is the one where the Tim protein pertainingto the present invention is bound on the carrier pertaining to thepresent invention.

Specifically, the Tim carrier of the present invention includes acarrier on which the Tim-1 protein pertaining to the present inventionis bound (hereafter it may be abbreviated as “the Tim-1 carrier of thepresent invention”, in some cases), a carrier on which the Tim-3 proteinpertaining to the present invention is bound (hereafter it may beabbreviated as “the Tim-3 carrier of the present invention”, in somecases), a carrier on which the Tim-4 protein pertaining to the presentinvention is bound (hereafter it may be abbreviated as “the Tim-4carrier of the present invention”, in some cases), or the like. Inaddition, a carrier on which two or more kinds of proteins selected fromthe Tim-1 protein pertaining to the present invention, Tim-3 proteinpertaining to the present invention, and Tim-4 protein pertaining to thepresent invention are bound is also encompassed in the Tim carrier ofthe present invention.

As the Tim carrier of the present invention, the Tim-4 carrier of thepresent invention is particularly preferable.

<Carrier Pertaining to the Present Invention>

As the carrier pertaining to the present invention, any carrier can beused as long as it is an insoluble carrier used in usual immunologicalassay, and includes an organic substance, such as, polystyrene,polyacrylic acid, polymethacrylic acid, polymethyl methacrylate,polyacryl amide, polyglycidyl methacrylate, polypropylene, polyolefin,polyimide, polyurethane, polyester, polyvinyl chloride, polyethylene,polychlorocarbonate, a silicone resin, silicone rubber, agarose,dextran, an ethylene-maleic anhydride copolymer, or the like; aninorganic substance, such as glass, silicon oxide, diatomaceous earth,porous glass, obscured glass, alumina, silica gel, a metal oxide, or thelike; a magnetic substance, such as iron, cobalt, nickel, magnetite,chromite, or the like; and those prepared using alloys of these magneticsubstances as raw materials. In addition, these carriers may be used ina wide variety of forms, such as a microplate, a tube, a disk-likepiece, particles (the beads), and the like.

It should be noted that it is preferable to use as particles (thebeads), when used in the obtaining method of the present invention, andthe removing method of the present invention, to be described later, andparticle size is not especially limited, however, there is included theone having usually 10 nm to 100 μm, and preferably 100 nm to 10 μm,according to objects and the applications.

In addition, the particles (the beads) or the microplate are preferablewhen used in the detection method of the present invention, to bedescribed later, and particle size is not especially limited andincludes the one having usually 10 nm to 100 μm, and preferably 100 nmto 10 μm, according to the objects and the applications. In addition,number and size of a well of the microplate are not especially limited,and include the one having usually 12 holes to 1536 holes, andpreferably 96 holes to 384 holes, according to the objects and theapplications.

<Binding Method for Tim Protein Pertaining to the Present Invention andCarrier Pertaining to the Present Invention>

The binding method for the Tim protein pertaining to the presentinvention and the carrier pertaining to the present invention may be inaccordance with a method known per se for binding a protein to acarrier, and the method includes, for example, a binding method byaffinity binding; a binding method by chemical binding (for example, amethod described in JP No. 3269554, WO 2012/039395); a binding method byphysical adsorption (for example, a method described in JP-B-05-41946),or the like, and the binding method by affinity binding and the bindingmethod by physical adsorption are preferable.

It should be noted that the binding method by affinity binding ispreferable, when used in the obtaining method and the removing method ofthe present invention to be described later. In addition, the bindingmethod by the affinity binding, or physical adsorption is preferable,when used in the detection method of the present invention, to bedescribed later.

<Binding Form of Tim Protein Pertaining to the Present Invention andCarrier Pertaining to the Present Invention>

Binding form of the Tim protein pertaining to the present invention andthe carrier pertaining to the present invention is not limited, as longas the carrier pertaining to the present invention and the Tim proteinpertaining to the present invention are bound, however, the one in whichthe carrier pertaining to the present invention binds to an SH group ofthe Tim protein pertaining to the present invention is preferable. Inaddition, the Tim protein pertaining to the present invention and thecarrier pertaining to the present invention may be bound either bydirect binding, or indirect binding via a chemical linker, an affinitysubstance [for example, a substance having affinity to an affinity-tag(to be described later), an antibody against the Tim protein pertainingto the present invention, biotins (to be described later), avidins (tobe described later), an antibody, or the like], or the like.

<Method for Binding by Affinity Binding>

The method for binding by affinity binding may be any one, as long as itis a method for binding by utilization of affinity binding (affinity)between substances, and includes, for example, the following (a) to (c):

(a) Method for Binding by Affinity Binding of Biotins and Avidins.

The Tim protein pertaining to the present invention and the carrierpertaining to the present invention can be bound, via the affinitysubstances, by using two or more kinds of substances (affinitysubstances) having affinity each other, comprising a combination of, forexample, biotins (biotin, iminobiotin, desthiobiotin, biocytin, biotinsulfoxide, and the like) and avidins (avidin, tamavidin, tamavidin 2,and the like), and the like.

It should be noted that either one of the affinity substances may bebound to the Tim protein pertaining to the present invention, and theremaining one may be bound to the carrier pertaining to the presentinvention, however, it is general, for example, that the avidins arebound to the carrier pertaining to the present invention, and biotinsare bound to the Tim protein pertaining to the present invention, whenbiotins and avidins are used.

(b) Method for Binding by Affinity Binding of the Affinity Tag and aSubstance Having Affinity to the Affinity Tag.

The Tim protein pertaining to the present invention and the carrierpertaining to the present invention can be bound, via the affinitysubstance by using the substance having affinity to the Tim proteinpertaining to the present invention (the affinity substance), forexample, the substance having affinity to the affinity tag (Protein A,Protein G, or the like).

It should be noted that the affinity substance is generally bound to thecarrier pertaining to the present invention.

(c) Method for Binding by Affinity Binding of the Antibody Against theTim Protein of the Present Invention and the Tim Protein of the PresentInvention.

The Tim protein pertaining to the present invention and the carrierpertaining to the present invention can be bound, via the affinitysubstance, by using the substance which has affinity to the Tim proteinpertaining to the present invention (the affinity substance), forexample, an antibody against the Tim protein pertaining to the presentinvention (an antibody against the affinity tag, such as an anti-FLAGtag antibody, an anti-His tag antibody, an anti-HA tag antibody, ananti-Myc tag antibody, an anti-MBP tag antibody, an anti-GST tagantibody, an anti-Strep (II) tag antibody, or the like; and an anti-Tim4 antibody (clone RMT 4-54) (produced by LifeSpan Biosciences Inc.), orthe like), or the like.

It should be noted that the affinity substance is generally bound to thecarrier pertaining to the present invention.

It should be noted that also as for a method for binding the Tim proteinpertaining to the present invention and/or the carrier pertaining to thepresent invention, and the affinity substance, in the above method, amethod for binding by physical adsorption, or a method for binding bychemical bonding, known per se, can be used, and the binding may becarried out directly or indirectly via a linker, or the like.

<Amount of Tim Protein Pertaining to the Present Invention to be Boundon Carrier Pertaining to the Present Invention>

Amount of the Tim protein pertaining to the present invention to bebound on the carrier pertaining to the present invention is, forexample, when the carrier pertaining to the present invention is thebeads, usually 0.1 μg to 50 μg, preferably 0.5 μg to 30 μg, and morepreferably 1.0 μg to 20 μg, relative to 1 mg of the carrier.

In addition, when the carrier pertaining to the present invention is themicroplate, it is usually 0.1 μg to 10 μg, preferably 0.2 μg to 5 μg,and more preferably 0.5 μg to 2 μg, relative to 1 well.

<Specific Preparation Methods for Tim Carrier of the Present Invention>

Explanation will be given below on specific preparation methods for theTim carrier of the present invention, taking the cases of preparation ofthe Tim carrier of the present invention, by the methods (a) to (c), asexamples.

<(a) Method for Binding by Affinity Binding of Biotins and Avidins>

First of all, in the method (a), the Tim protein pertaining to thepresent invention is prepared, according to the preparation method forthe Tim protein pertaining to the present invention. Next, the Timprotein pertaining to the present invention-biotins complex is formed bybinding the Tim protein pertaining to the present invention and biotins(hereinafter, it may be abbreviated as “biotin labelling” or“biotinylation”, in some cases). On the other hand, the carrierpertaining to the present invention-avidins complex is formed, bybinding avidins to the carrier pertaining to the present invention(hereinafter, it may be abbreviated as “the carrier pertaining to thepresent invention bound with avidins”, in some cases). The Tim carrierof the present invention is obtained, by bringing into contacted theresulting Tim protein pertaining to the present invention-biotinscomplex, and the carrier pertaining to the present invention-avidinscomplex, to bind biotins in the Tim protein pertaining to the presentinvention-biotins complex, and avidins in the carrier pertaining to thepresent invention-avidins complex. —Binding of Tim Protein Pertaining tothe Present Invention and Biotins (Biotin Labelling)—

In the method (a), binding of the Tim protein pertaining to the presentinvention and biotins may be carried out by using a commerciallyavailable biotin labelling-kit, or according to a usual method of biotinlabelling of a protein, by appropriately adjusting reagents required. Amethod for using the commercially available biotin labelling-kit of theprotein (Biotinylation kit) may be in accordance with a method describedin a protocol attached to Biotin Labeling Kit-SH (manufactured byDojindo Molecular Technologies, Inc.), or Biotin Labeling Kit-NH₂(manufactured by Dojindo Molecular Technologies, Inc.).

Amount of biotins to be bound with 1 μg of the Tim protein pertaining tothe present invention is usually 10 ng to 1.0 μg, preferably 20 ng to200 ng, and more preferably 30 ng to 150 ng.

A site for binding biotins to the Tim protein pertaining to the presentinvention is preferably an SH group of the Tim protein pertaining to thepresent invention.

—Carrier Pertaining to the Present Invention-Avidins Complex (Carrier ofthe Present Invention Bound with Avidins)—

As the carrier pertaining to the present invention-avidins complex (thecarrier pertaining to the present invention bound with avidins), in themethod (a), commercially available one may be used, or it may beprepared in accordance with a usual method by appropriately adjustingreagents required. The carrier pertaining to the presentinvention-avidins complex includes, for example, the beads or themicroplate bound with avidin; the beads or the microplate bound withtamavidin; the beads or the microplate bound with tamavidin 2; the beadsor the microplate bound with streptavidin, or the like; and thecommercially available one includes Dynabeads M-270 Streptavidin C1(manufactured by Thermo Fisher Scientific, Inc.), FGbeads Streptavidin(manufactured by Tamagawa Seiki Co., Ltd.), Avidin plate (manufacturedby Sumitom Bakelite Co., Ltd.), or the like.

Amount of avidins to be brought into contacted with 1 mg of the carrierpertaining to the present invention is usually 5.0 to 150 μg, preferably10 to 100 μg, and more preferably 20 to 50 μg, when the carrierpertaining to the present invention is, for example, the beads, inbinding of the carrier pertaining to the present invention and avidins.Amount of avidins to be brought into contacted to 1 well is usually 0.1μg to 10 μg, preferably 0.2 μg to 5 μg, and more preferably 0.5 μg to 2μg, when the carrier pertaining to the present invention is, forexample, the microplate. —Binding of Carrier Pertaining to the PresentInvention-Avidins Complex, and Tim Protein Pertaining to the PresentInvention-Biotins Complex—

To obtain the Tim carrier of the present invention (by binding thecarrier pertaining to the present invention-avidins complex, and the Timprotein pertaining to the present invention-biotins complex), in themethod (a), usually 0.1 mg to 10 mg, preferably 0.3 mg to 5.0 mg, andmore preferably 0.5 to 3.0 mg of the carrier pertaining to the presentinvention-avidins complex, and usually 1.0 to 50 μg, preferably 1.0 to30 μg, and more preferably 1.0 to 20 μg of the Tim protein pertaining tothe present invention-biotins complex, relative to 1 mg of the carrierpertaining to the present invention-avidins complex, may be brought intocontacted, when the carrier pertaining to the present invention is, forexample, the beads; in addition, the carrier pertaining to the presentinvention-avidins complex, relative to 1 well, and usually 1.0 to 10 μg,preferably 1.0 to 5.0 μg, and more preferably 1.0 to 2.0 μg of the Timprotein pertaining to the present invention-biotins complex may bebrought into contacted, when the carrier pertaining to the presentinvention is the microplate, and then subjected to a reaction at usually4.0° C. to 37° C., preferably 11° C. to 30° C., and more preferably 20°C. to 25° C., for usually 0.5 hour to 24 hours, preferably 0.5 hour to8.0 hours, and more preferably 0.5 hour to 2.0 hours to bind avidins andbiotins. In this way, the carrier pertaining to the presentinvention-avidins complex, and the Tim protein pertaining to the presentinvention-biotins complex are bound to obtain the Tim carrier of thepresent invention.

It should be noted that contact of the carrier pertaining to the presentinvention-avidins complex, and the Tim protein pertaining to the presentinvention-avidins complex is generally carried out by bringing intocontacted a solution containing the Tim protein pertaining to thepresent invention-biotins complex, and the carrier pertaining to thepresent invention-avidins complex.

The solution for containing the Tim protein pertaining to the presentinvention-biotins complex may be any solution, as long as it is capableof dissolving the Tim protein pertaining to the presentinvention-biotins complex in a stable state, and includes for example,purified water, a buffer solution which has buffer action, for example,at pH 7.0 to 8.0, and preferably 7.2 to 7.6 (for example, PBS, TBS, HBSor the like). In addition, buffer agent concentration in these buffersolutions is appropriately selected from a range of usually 5.0 to 50mM, and preferably 10 to 30 mM, and NaCl concentration is appropriatelyselected from a range of usually 100 to 200 mM, and preferably 140 to160 mM. In addition, for example, saccharides, salts, such as NaCl, andthe like, a surfactant, a preservative, a protein, or the like, may becontained in this solution, as long as in such an amount that does notinhibit binding of the carrier pertaining to the presentinvention-avidins complex, and the Tim protein pertaining to the presentinvention-biotins complex, after contacting the carrier pertaining tothe present invention-avidins complex, and the solution containing theTim protein pertaining to the present invention-biotins complex. Thesurfactant includes, for example, Tween 20, or the like, and surfactantconcentration in the solution for containing the Tim protein pertainingto the present invention-biotins complex is usually 0.00001 to 0.2%, andpreferably 0.0005 to 0.1%. It should be noted that the solutiondissolving the Tim protein pertaining to the present invention-biotinscomplex in these solvents may be abbreviated as “the Tim proteinpertaining to the present invention-biotins complex-containingsolution”, in some cases.

—Specific Preparation Method for Tim Carrier of the Present Invention byMethod (a)—

A specific preparation method for the Tim carrier of the presentinvention by the method (a) may be carried out, for example, by thefollowing method.

First of all, the Tim protein pertaining to the present invention isprepared, according to the preparation method for the Tim proteinpertaining to the present invention. Next, the Tim protein pertaining tothe present invention-biotins complex is formed by binding biotins tothe Tim protein pertaining to the present invention, according to anattached protocol in the Biotin Labeling Kit-SH (manufactured by DojindoMolecular Technologies, Inc.), the Biotin Labeling Kit-NH₂ (manufacturedby Dojindo Molecular Technologies, Inc.), or a usual method in biotinlabelling of the protein.

After that, when the carrier pertaining to the present invention is thebeads, the Tim carrier of the present invention is obtained, by bringinginto contacted usually 0.1 mg to 10 mg, preferably 0.3 mg to 5.0 mg, andmore preferably 0.5 to 3.0 mg of the carrier pertaining to the presentinvention-avidins complex, and usually 50 μL to 1500 μL, preferably 100μL to 1000 μL, and more preferably 200 μL to 500 μL of a solutioncontaining usually 1.0 to 50 μg, preferably 1.0 to 30 μg, and morepreferably 1.0 to 20 μg of the Tim protein pertaining to the presentinvention-biotins complex (a solution containing the Tim proteinpertaining to the present invention-biotins complex, for example, inpurified water, or in buffer solution, or the like, which has bufferaction at pH 7.0 to 8.0, and preferably 7.2 to 7.6), relative to 1 mg ofthe carrier pertaining to the present invention-avidins complex, andthen subjecting them to a reaction at usually 4.0° C. to 37° C.,preferably 11° C. to 30° C., and more preferably 20° C. to 25° C., forusually 0.5 hour to 24 hours, preferably 0.5 hour to 8.0 hours, and morepreferably 0.5 hour to 2.0 hours, to bind avidins in the carrierpertaining to the present invention-avidins complex, and biotins in theTim protein pertaining to the present invention-biotins complex.

When the carrier pertaining to the present invention is the microplate,the Tim carrier of the present invention is obtained by bringing intocontacted the carrier pertaining to the present invention-avidinscomplex, relative to 1 well, and usually 50 μL to 300 μL, preferably 50μL to 200 μL, more preferably 100 μL to 200 μL of a solution containingusually 1.0 to 10 μg, preferably 1.0 to 5.0 μg, and more preferably 1.0to 2.0 μg of the Tim protein pertaining to the present invention-biotinscomplex (a solution containing the Tim protein pertaining to the presentinvention-biotins complex, for example, in purified water, or in abuffer solution, or the like, which has buffer action at pH 7.0 to 8.0,and preferably 7.2 to 7.6), and then subjecting them to a reaction atusually 4.0° C. to 37° C., preferably 11° C. to 30° C., and morepreferably 20° C. to 25° C., usually for 0.5 hour to 24 hours,preferably 0.5 hour to 8.0 hours, and more preferably 0.5 hour to 2.0hours, to bind avidins in the carrier pertaining to the presentinvention-avidins complex, and biotins in the Tim protein pertaining tothe present invention-biotins complex.

<(b) Method for Binding by Affinity Binding of Affinity-Tag, andSubstance Having Affinity to Affinity-Tag>

In the method of (b), first of all, the Tim protein pertaining to thepresent invention having the affinity-tag is prepared, according to thepreparation method for the Tim protein pertaining to the presentinvention. On the other hand, the carrier pertaining to the presentinvention-the affinity substance complex (hereinafter, it may beabbreviated as “the carrier pertaining to the present invention boundwith the affinity substance”, in some cases) is formed, by binding thesubstance having affinity to the affinity-tag (the affinity substance)to the carrier pertaining to the present invention. The Tim carrier ofthe present invention is obtained by bringing into contacted theresulting carrier pertaining to the present invention-affinity substancecomplex, and the Tim protein pertaining to the present invention havingthe affinity-tag to bind the affinity substance in the carrierpertaining to the present invention-the affinity substance complex, andthe affinity-tag in the Tim protein pertaining to the present inventionhaving the affinity-tag, and to bind the carrier pertaining to thepresent invention-affinity substance complex, and the Tim proteinpertaining to the present invention having the affinity-tag.

—Carrier Pertaining to the Present Invention-Affinity Substance Complex—

As the carrier pertaining to the present invention-the affinitysubstance complex in the method (b), the commercially available one maybe used, or it may be prepared, according to a usual method byappropriately adjusting reagents required. The carrier pertaining to thepresent invention-the affinity substance complex includes, for example,the beads or the microplate bound with Protein G, the beads or themicroplate bound with Protein A, or the like, and the commerciallyavailable one includes, Dynabeads Protein G (manufactured by ThermoFisher Scientific Co.), Dynabeads Protein A (manufactured by ThermoFisher Scientific Co.), FG beads Protein G (manufactured byTamagawaseiki Co. Ltd.), FG beads Protein A (manufactured byTamagawaseiki Co. Ltd.), or the like.

Amount of the affinity substance to be brought into contacted with 1 mgof the carrier pertaining to the present invention, in binding of thecarrier pertaining to the present invention and the affinity substance,is usually 5.0 to 50 μg, preferably 10 to 50 μg, and more preferably 20to 50 μg, when the carrier pertaining to the present invention is thebeads. Amount of the affinity substance to be brought into contactedwith 1 well is usually 0.1 μg to 10 μg, preferably 0.2 μg to 5 μg, andmore preferably 0.5 μg to 2 μg, when the carrier pertaining to thepresent invention is the microplate.

—Binding of Carrier Pertaining to the Present Invention-AffinitySubstance Complex, and Tim Protein Pertaining to the Present InventionHaving Affinity-Tag—

In the method (b), for example, the following method may be carried outto obtain the Tim carrier of the present invention (by binding thecarrier pertaining to the present invention-affinity substance complex,and the Tim protein pertaining to the present invention having theaffinity-tag). The Tim carrier of the present invention is obtained bybringing into contacted usually 0.1 mg to 10 mg, preferably 0.3 mg to5.0 mg, and more preferably 0.5 to 3.0 mg of the carrier pertaining tothe present invention-affinity substance complex, and usually 1.0 to 50μg, preferably 1.0 to 30 μg, and more preferably 1.0 to 20 μg of the Timprotein pertaining to the present invention having the affinity-tag,relative to 1 mg of the carrier pertaining to the presentinvention-affinity substance complex, when the carrier pertaining to thepresent invention is the beads; and by bringing into contacted thecarrier pertaining to the present invention-affinity substance complex,relative to 1 well, and usually 1.0 to 10 μg, preferably, 1.0 to 5.0 μg,and more preferably 1.0 to 2.0 μg of the Tim protein pertaining to thepresent invention having the affinity-tag, when the carrier pertainingto the present invention is the microplate, and by subjecting them to areaction at usually 4.0° C. to 37° C., preferably 11° C. to 30° C., morepreferably 20° C. to 25° C., for usually 0.5 hour to 24 hours,preferably 0.5 hour to 8.0 hours, more preferably 0.5 hour to 2.0 hours,to bind the carrier pertaining to the present invention-affinitysubstance complex, and the Tim protein pertaining to the presentinvention having the affinity-tag.

It should be noted that contact of the carrier pertaining to the presentinvention-the affinity substance complex, and the Tim protein pertainingto the present invention having the affinity-tag, in the method (b), isgenerally carried out by bringing into contacted the carrier pertainingto the present invention-the affinity substance complex, and a solutioncontaining the Tim protein pertaining to the present invention havingthe affinity-tag.

The solution for containing the Tim protein pertaining to the presentinvention having the affinity tag may be any solution, as long ascapable of dissolving the Tim protein pertaining to the presentinvention having the affinity-tag, in a stable state, and includes forexample, purified water, or a buffer solution which has buffer action,for example, at pH 7.0 to 8.0, and preferably 7.2 to 7.6 (for example,PBS, TBS, HBS or the like). In addition, buffer agent concentration inthese buffer solutions is appropriately selected from a range of usually5.0 to 50 mM, and preferably 10 to 30 mM, and NaCl concentration isappropriately selected from a range of usually 100 to 200 mM, andpreferably 140 to 160 mM.

In addition, for example, saccharides, salts, such as NaCl, and thelike, a surfactant, a preservative, a protein, or the like, may becontained in this solution, as long as in such an amount that does notinhibit binding of the Tim protein pertaining to the present inventionhaving the affinity-tag, and the carrier pertaining to the presentinvention-the affinity-substance complex, after contacting the solutioncontaining the Tim protein pertaining to the present invention havingthe affinity-tag, and the carrier pertaining to the presentinvention-the affinity substance complex. The surfactant includes, forexample, Tween 20, or the like, and surfactant concentration in thesolution for containing the Tim protein pertaining to the presentinvention having the affinity-tag, is usually 0.00001 to 0.2%, andpreferably 0.0005 to 0.1%. It should be noted that the solutiondissolving the Tim protein pertaining to the present invention havingthe affinity-tag, in these solutions, may be abbreviated as “the Timprotein pertaining to the present invention having theaffinity-tag-containing solution”, in some cases.

—Specific Preparation Method for Tim Carrier of the Present Invention byMethod (b)—

A specific preparation method for the Tim carrier of the presentinvention in the method (b) may be carried out, for example, by thefollowing method.

First of all, the Tim protein pertaining to the present invention havingthe affinity-tag is prepared, according to the preparation method forthe Tim protein pertaining to the present invention.

Next, when the carrier pertaining to the present invention is the beads,the Tim carrier of the present invention is obtained by bringing intocontacted usually 0.1 mg to 10 mg, preferably 0.3 mg to 5.0 mg, and morepreferably 0.5 to 3.0 mg of the carrier pertaining to the presentinvention-the affinity substance complex, and usually 50 μL to 1500 μL,preferably 100 μL to 1000 μL, and more preferably 200 μL to 500 μL ofthe solution containing usually 1.0 to 50 μg, preferably 1.0 to 30 μg,and more preferably 1.0 to 20 μg of the Tim protein pertaining to thepresent invention having the affinity-tag (the solution containing theTim protein pertaining to the present invention having the affinity-tag,for example, in purified water, or in the buffer solution at pH 7.0 to8.0, or the like), relative to 1 mg of the carrier pertaining to thepresent invention-the affinity substance complex, and are subjected themto a reaction at usually 4.0° C. to 37° C., preferably 11° C. to 30° C.,and more preferably 20° C. to 25° C., for usually 0.5 hour to 24 hours,preferably 0.5 hour to 8.0 hours, and more preferably 0.5 hour to 2.0hours, to bind the affinity substance in the carrier pertaining to thepresent invention-the affinity substance complex, and the affinity-tagin the Tim protein pertaining to the present invention having theaffinity-tag.

When the carrier pertaining to the present invention is the microplate,the Tim carrier of the present invention is obtained by bringing intocontacted the carrier pertaining to the present invention-the affinitysubstance complex, relative to 1 well, and usually 50 μL to 300 μL,preferably 50 μL to 200 μL, and more preferably 100 μL to 200 μL of thesolution containing 1.0 to 10 μg, preferably 1.0 to 5.0 μg, and morepreferably 1.0 to 2.0 μg of the Tim protein pertaining to the presentinvention having the affinity-tag pertaining to the present invention(the solution containing the Tim protein pertaining to the presentinvention-biotins complex, for example, in purified water, or the buffersolution, or the like, which has buffer action at pH 7.0 to 8.0, andpreferably 7.2 to 7.6), and are subjected them to a reaction at usually4.0° C. to 37° C., preferably 11° C. to 30° C., and more preferably 20°C. to 25° C., for usually 0.5 hour to 24 hours, preferably 0.5 hour to8.0 hours, and more preferably 0.5 hour to 2.0 hours, to bind theaffinity substance in the carrier pertaining to the presentinvention-the affinity substance complex, and the affinity-tag in theTim protein pertaining to the present invention having the affinity-tag.

<(C) Method for Binding by Affinity Binding of Antibody Against TimProtein of the Present Invention and Tim Protein of the PresentInvention>

First of all, the Tim protein pertaining to the present invention isprepared, according to the preparation method for the Tim proteinpertaining to the present invention. On the other hand, an antibodyagainst the Tim protein pertaining to the present invention (hereinafterit may be abbreviated as “the anti-Tim antibody”, in some cases) isbound to the carrier pertaining to the present invention to form acarrier pertaining to the present invention-anti-Tim antibody complex.The Tim carrier of the present invention is obtained by bringing intocontacted the resulting carrier pertaining to the presentinvention-anti-Tim antibody complex, and the Tim protein pertaining tothe present invention to bind the antibody against the Tim protein inthe carrier pertaining to the present invention-anti-Tim antibodycomplex, and the Tim protein pertaining to the present invention.

It should be noted that, in the above description, when the Tim proteinhaving the affinity tag (a protein having the amino acid sequence of theTim protein and the amino acid sequence of the affinity tag) is used asthe Tim protein pertaining to the present invention, as the anti-Timantibody, either an antibody recognizing the Tim protein (an antibodyrecognizing a protein portion, based on the amino acid sequence of theTim protein), or an antibody recognizing the affinity tag (an antibodyrecognizing a protein portion, based on the amino acid sequence of theaffinity tag) may be used. In addition, when the Tim protein not havingthe affinity tag (a protein consisting only of the amino acid sequenceof the Tim protein) is used as the Tim protein pertaining to the presentinvention, as the anti-Tim antibody, an antibody that recognizes the Timprotein (an antibody that recognizes a protein portion, based on theamino acid sequence of the Tim protein) may be used.

—Carrier Pertaining to the Present Invention-Anti-Tim Antibody Complex(the Carrier Pertaining to the Present Invention to which Antibody isBound)—

In the method (c), as the carrier pertaining to the presentinvention-anti-Tim antibody complex, a commercially available one may beused, or it may be prepared, according to a usual method by arrangingnecessary reagents appropriately. The carrier pertaining to the presentinvention-anti-Tim antibody complex includes, for example, the beads orthe microplates to which an anti-Fc tag antibody is bound, the beads orthe microplates to which an anti-FLAG tag antibody is bound, the beadsor the microplates to which an anti-His tag antibody is bound, the beadsor the microplates to which an anti-GST tag is bound, the beads or themicroplates to which an anti-MBP tag is bound, the beads or themicroplates to which an anti-HA tag is bound, the beads or themicroplates to which an anti-Myc tag is bound, the beads or themicroplates to which an anti-Strep (II) tag is bound, the beads or themicroplates to which an antibody against an anti-Tim protein is bound,or the like, and as a commercially available one, the anti-DYKDDDDK tagantibody magnetic beads (produced by Wako Pure Chemical Industries,Ltd.), or the like, is included.

The origin of the anti-Tim antibody is not especially limited. Inaddition, either polyclonal antibodies or monoclonal antibodies may beused, and the monoclonal antibodies are preferable. In addition, as theanti-Tim antibody, a commercially available one may be used, or it maybe prepared according to a usual method by arranging necessary reagentsappropriately. Specific examples of the anti-Tim antibody are asdescribed above.

Amount of the antibody, relative to the Tim protein pertaining to thepresent invention, to be brought into contacted with 1 mg of the carrierpertaining to the present invention, is usually 5.0 to 50 μg, preferably10 to 50 μg, and more preferably 20 μg to 50 μg, when the obtainingmethod of the present invention and the removing method of the presentinvention are used, when the carrier pertaining to the present inventionis the beads, in binding of the carrier pertaining to the presentinvention and the anti-Tim antibody. When the carrier pertaining to thepresent invention is the microplate, it is usually 0.1 μg to 10 μg,preferably 0.2 μg to 5 μg, and more preferably 0.5 μg to 2 μg, relativeto 1 well.

—Binding of Carrier Pertaining to the Present Invention-Anti-TimAntibody Complex and Tim Protein Pertaining to the Present Invention

A method for obtaining the Tim carrier of the present invention, bybinding of the carrier pertaining to the present invention-the anti-Timantibody complex, and the Tim protein pertaining to the presentinvention, in the method (c), includes, for example, the followingmethods. That is, when the carrier pertaining to the present inventionis the beads, the Tim carrier of the present invention is obtained bybringing into contacted usually 0.1 mg to 10 mg, preferably 0.3 mg to5.0 mg, and more preferably 0.5 to 3.0 mg of the carrier pertaining tothe present invention-the anti-Tim antibody complex, and usually 1.0 to50 μg, preferably 1.0 to 30 μg, and more preferably 1.0 to 20 μg of theTim protein pertaining to the present invention, relative to 1 mg of thecarrier pertaining to the present invention-the anti-Tim antibodycomplex; and when the carrier pertaining to the present invention is themicroplate, by bringing into contacted the carrier pertaining to thepresent invention-the anti-Tim antibody complex, relative to 1 well, andusually 1.0 to 10 μg, preferably 1.0 to 5.0 μg, and more preferably 1.0to 2.0 μg of the Tim protein pertaining to the present invention, and bysubjecting them to a reaction at usually 4.0° C. to 37° C., preferably11° C. to 30° C., and more preferably 20° C. to 25° C., for usually 0.5hour to 24 hours, preferably 0.5 hour to 8.0 hours, and more preferably0.5 hour to 2.0 hours to bind the anti-Tim antibody in the carrierpertaining to the present invention-the anti-Tim antibody complex, andthe Tim protein pertaining to the present invention.

It should be noted that contact between the carrier pertaining to thepresent invention-the anti-Tim antibody complex, and the Tim proteinpertaining to the present invention, in the method (c), is carried outgenerally by bringing into contacted the carrier pertaining to thepresent invention-the anti-Tim antibody complex, and a solutioncontaining the Tim protein pertaining to the present invention.

The solution for containing the Tim protein pertaining to the presentinvention may be any one, as long as it dissolves the Tim proteinpertaining to the present invention in a stable state, and includes, forexample, purified water, or the buffer solution which has buffer action,for example, at pH 7.0 to 8.0, and preferably 7.2 to 7.6 (for example,PBS, TBS, HBC or the like). In addition, buffer agent concentration inthese buffer solutions is appropriately selected from a range of usually5 to 50 mM, and preferably 10 to 30 mM, and NaCl concentration isappropriately selected from a range of usually 100 to 200 mM, andpreferably 140 to 160 mM. In addition, for example, saccharides, salts,such as NaCl, and the like, a surfactant, a preservative, a protein, orthe like, may be contained in this solution, as long as in such anamount that does not inhibit binding of the Tim protein pertaining tothe present invention and the carrier pertaining to the presentinvention-the anti-Tim antibody complex, after contacting the solutioncontaining to Tim protein pertaining to the present invention, and thecarrier pertaining to the present invention-the anti-Tim antibodycomplex. The surfactant includes, for example, Tween 20, or the like,and surfactant concentration in the solution containing the Tim proteinpertaining to the present invention is usually 0.00001 to 0.2%, andpreferably 0.0005 to 0.1%. It should be noted that the solutiondissolving the Tim protein pertaining to the present invention in thesesolutions may be abbreviated as “the Tim protein pertaining to thepresent invention-containing solution”, in some cases.

—Specific Preparation Method for Tim Carrier of the Present Invention byMethod (c)—

A specific preparation method for the Tim carrier of the presentinvention in the method (c) may be carried out, for example, by thefollowing method.

First of all, the Tim protein pertaining to the present invention isprepared, according to the preparation method for the Tim proteinpertaining to the present invention. Next, when the carrier pertainingto the present invention is the beads, the Tim carrier of the presentinvention is obtained, by bringing into contacted usually 0.1 mg to 10mg, preferably 0.3 mg to 5.0 mg, and more preferably 0.5 mg to 3.0 mg ofthe carrier pertaining to the present invention-the anti-Tim antibodycomplex, and usually 100 to 1000 μL, and preferably 200 to 500 μL of thesolution containing usually 1.0 to 50 μg, preferably 1.0 to 30 μg, andmore preferably 1.0 to 20 μg of the Tim protein pertaining to thepresent invention, relative to 1 mg of the carrier pertaining to thepresent invention-anti-Tim antibody complex (for example, the solutioncontaining the Tim protein pertaining to the present invention, forexample, in purified water, or the buffer solution, for example, at pH7.0 to 8.0), and by subjecting them to a reaction at usually 4° C. to37° C., preferably 11° C. to 30° C., and more preferably 20° C. to 25°C., for usually 0.5 hour to 24 hours, preferably 0.5 hour to 8.0 hours,and more preferably 0.5 hour to 2.0 hours to bind the antibody againstthe Tim protein pertaining to the present invention in the carrierpertaining to the present invention-the anti-Tim antibody complex, andthe Tim protein pertaining to the present invention. When the carrierpertaining to the present invention is the microplate, the Tim carrierof the present invention is obtained by bringing into contacted thecarrier pertaining to the present invention-the anti-Tim antibodycomplex, relative to 1 well, and usually 50 μL to 300 μL, preferably 50μL to 200 μL, and more preferably 100 μL to 200 μL of the solutioncontaining 1.0 to 10 μg, preferably 1.0 to 5.0 μg, and more preferably1.0 to 2.0 μg of the Tim protein pertaining to the present invention(the solution containing the Tim protein pertaining to the presentinvention-biotins complex, for example, in purified water, or the buffersolution having buffer action at pH 7.0 to 8.0, and preferably 7.2 to7.6), and by subjecting them to a reaction at usually 4.0° C. to 37° C.,preferably 11° C. to 30° C., and more preferably 20° C. to 25° C., forusually 0.5 hour to 24 hours, preferably 0.5 hour to 8.0 hours, and morepreferably 0.5 hour to 2.0 hours to bind the affinity substance in thecarrier pertaining to the present invention-the affinity substancecomplex, and the affinity-tag in the Tim protein pertaining to thepresent invention having the affinity-tag.

<Method for Binding by Physical Adsorption>

As the method for binding the Tim protein pertaining to the presentinvention and the carrier pertaining to the present invention byphysical adsorption, the Tim protein pertaining to the present inventionand the carrier pertaining to the present invention may be brought intocontacted under condition where the Tim protein pertaining to thepresent invention and the carrier pertaining to the present inventionbind, according to a known method per se.

Reaction temperature of the Tim protein pertaining to the presentinvention and the carrier pertaining to the present invention is usually2° C. to 37° C., and preferably 4° C. to 11° C.

Reaction time of the Tim protein pertaining to the present invention andthe carrier pertaining to the present invention is usually 4 hours to 48hours, and preferably 12 hours to 24 hours.

Amount of the Tim protein pertaining to the present invention to bebrought into contacted with 1 mg of the carrier pertaining to thepresent invention is usually 5.0 to 50 μg, preferably 10 to 50 μg, andmore preferably 20 to 50 μg, when the carrier pertaining to the presentinvention is the beads. Amount of the Tim protein pertaining to thepresent invention to be brought into contacted with 1 well is usually0.1 μg to 10 μg, preferably 0.2 μg to 5 μg, and more preferably 0.5 μgto 2 μg, when the carrier pertaining to the present invention is themicroplate.

It should be noted that physical adsorption of the Tim proteinpertaining to the present invention and the carrier pertaining to thepresent invention may be carried out generally by bringing intocontacted the solution containing the Tim protein pertaining to thepresent invention, and the carrier pertaining to the present invention.

The solution for containing the Tim protein pertaining to the presentinvention may be any solution, as long as being capable of dissolvingthe Tim protein pertaining to the present invention in a stable state,and includes for example, purified water, or a buffer solution which hasbuffer action, for example, at pH 6.0 to 9.5, and preferably 7.0 to 8.0(for example, a Good's buffer solution, such as MOPS, or the like, acarbonate buffer solution, PBS, TBS, HBS or the like). In addition,buffer agent concentration in these buffer solutions is appropriatelyselected from a range of usually 5 to 100 mM, and preferably 10 to 50mM, and NaCl concentration, if added, is appropriately selected from arange of usually 100 to 200 mM, and preferably 140 to 160 mM. Inaddition, for example, saccharides, salts, such as NaCl, and the like, asurfactant, a preservative, a protein, or the like, may be contained inthis solution, as long as in such an amount that does not inhibitbinding of the carrier of the present invention and the extracellularmembrane vesicles or the viruses, after contacting the solutioncontaining the Tim protein pertaining to the present invention, and thecarrier pertaining to the present invention. It should be noted that thesolution dissolving the Tim protein pertaining to the present inventionin these solutions may be abbreviated as “the Tim protein pertaining tothe present invention-containing solution”, in some cases.

Specific examples of the binding method by physical adsorption, in thebinding method for the Tim protein pertaining to the present inventionand the carrier pertaining to the present invention, include, forexample, the following methods.

First of all, the Tim protein pertaining to the present invention isprepared, according to a preparation method for the Tim proteinpertaining to the present invention. Next, the Tim carrier of thepresent invention is obtained by bringing into contacted usually 50 μLto 300 μL, preferably 50 μL to 200 μL, and more preferably 50 μL to 100μL of the solution containing usually 5.0 to 50 μg, preferably 10 to 50μg, and more preferably 20 to 50 μg of the Tim protein (the solutioncontaining the Tim protein pertaining to the present invention, forexample, in purified water, or in a buffer solution of, for example, pH7.0 to 8.0, or the like), to 1 mg of the carrier pertaining to thepresent invention when the carrier pertaining to the present inventionis the beads; or by bringing into contacted usually 50 μL to 300 μL,preferably 50 μL to 200 μL, and more preferably 50 μL to 100 μL of asolution containing usually 0.1 μg to 10 μg, preferably 0.2 μg to 5 μg,and more preferably 0.5 μg to 2 μg of the Tim protein (the solutioncontaining the Tim protein pertaining to the present invention, forexample, in purified water, or in a buffer solution of, for example, pH7.0 to 8.0, or the like), to 1 well when the carrier pertaining to thepresent invention is the microplate; and by subjecting them to areaction at usually 2° C. to 37° C., and preferably 4° C. to 11° C., forusually 4 hours to 48 hours, and preferably 12 hours to 24 hours to bindthe carrier pertaining to the present invention and the Tim proteinpertaining to the present invention.

<Treatment of Tim Carrier of the Present Invention>

The resulting Tim carrier of the present invention as above may besubjected to blocking treatment usually carried out in this field.

The resulting Tim carrier of the present invention as above may besubjected to purification treatment usually carried out in this field,as needed. Purification treatment is not especially limited, as long asimpurities adhered on the carrier surface can be removed, and includes,for example, a method for washing the Tim carrier of the presentinvention with a washing solution (hereafter it may be abbreviated as“washing operation”, in some cases).

Explanation will be given on an example by taking the case of using themagnetic particles, as the carrier pertaining to the present invention.

First of all, a container, containing the solution containing theresulting Tim carrier of the present invention as described above, isinstalled in a magnet stand, to collect the Tim carrier of the presentinvention on a tube wall using magnetic force, and the solution in thecontainer is discarded. Next, the washing solution is added in thecontainer, and stirred. After that, the container is installed in themagnet stand, the same as the above, to collect the Tim carrier of thepresent invention on the tube wall by using magnetic force, and thesolution in the container is discarded. The washing operation may berepeated several times, as necessary. The washing solution to be used inthe washing operation may be any solution, as long as it does not affectbinding of the Tim protein pertaining to the present invention in theTim carrier of the present invention, and the carrier of the presentinvention, and includes, for example, purified water or the buffersolution which has buffer action, for example, at pH 7.0 to 8.0, andpreferably 7.2 to 7.6 (for example, PBS, TBS, HBC, or the like). Inaddition, buffer agent concentration in these solutions is appropriatelyselected from a range of usually 5 to 50 mM, and preferably 10 to 30 mM,and NaCl concentration is appropriately selected from a range of usually100 to 200 mM, and preferably 140 to 160 mM. This solution may contain,for example, saccharides, salts, such as NaCl, and the like, asurfactant, a preservative, a protein, or the like, as long as in suchan amount that does not inhibit binding of the Tim protein pertaining tothe present invention and the carrier pertaining to the presentinvention.

The surfactant includes, for example, Tween 20, or the like, andsurfactant concentration in the washing solution is usually 0.00001 to0.2%, and preferably 0.0005 to 0.1%.

The extracellular membrane vesicles or the viruses can be obtainedefficiently, and the extracellular membrane vesicles or the viruses inthe sample can be obtained in high purity, by using the Tim carrier ofthe present invention. In addition, the extracellular membrane vesiclesor the viruses in the sample can also be removed efficiently. Further,the extracellular membrane vesicles or the viruses in the sample canalso be detected in high sensitivity.

<6. Obtaining Method for Extracellular Membrane Vesicles or the Virusesin Sample>

A method for obtaining the extracellular membrane vesicle or the virusof the present invention (hereafter it may be abbreviated as “theobtaining method of the present invention”, in some cases) ischaracterized by comprising the following steps:

(1) A step for forming a complex of the Tim protein bound to thecarrier, and the extracellular membrane vesicle or the virus in thesample, in the presence of the calcium ion (hereafter it may beabbreviated as “the complex formation step”, in some cases).(2) A step for separating the complex and the sample (hereafter it maybe abbreviated as “the complex separation step”, in some cases).(3) A step for separating the extracellular membrane vesicle or thevirus from the complex to obtain the extracellular membrane vesicles orthe viruses (hereafter it may be abbreviated as “the obtaining step”, insome cases).<6-1. Complex Formation Step>

The complex formation step is a step for forming the complex of the Timprotein, the carrier, and the extracellular membrane vesicle or thevirus in the sample, in the presence of the calcium ion.

<Sample Pertaining to the Present Invention>

The sample pertaining to the present invention may be either of the onecontaining the extracellular membrane vesicle or the virus pertaining tothe present invention in liquid, or the one possibly containing theextracellular membrane vesicle or the virus pertaining to the presentinvention. The sample pertaining to the present invention may be eitherof the one derived from a living organism or may be the one where theextracellular membrane vesicle or the virus pertaining to the presentinvention is dissolved or suspended in a solution, such as a medium or abuffer solution, or the like. Specific examples of the sample pertainingto the present invention include body fluids, such as blood, saliva,urine, milk, amniotic fluid, ascites, and the like, cell culturesupernatant, and the like.

The solution for containing (dissolving or suspending) the extracellularmembrane vesicle or the virus pertaining to the present invention may beany one, as long as it dissolves or suspends the extracellular membranevesicle or the virus pertaining to the present invention in a stablestate, and does not inhibit binding of the complex of the Tim proteinbound to the carrier, and the extracellular membrane vesicle or thevirus in the sample, and includes, for example, water, a buffer solutionhaving buffering action at pH 7.0 to pH 8.0, and preferably pH 7.2 to pH7.6 (for example, TBS, HBS, or the like), or the like. It should benoted that, a phosphate buffer is not preferable because it binds withcalcium and causes precipitation. In addition, concentration of thebuffering agents in these buffer solutions is appropriately selectedusually from a range of 5 to 50 mM, and preferably 10 to 30 mM, and NaClconcentration is appropriately selected from a range of usually 100 to200 mM, and preferably 140 to 160 mM.

This solution may contain, for example, saccharides, salts, such asNaCl, and the like, a surfactant, an antiseptic agent, a protein, or thelike, as long as in such an amount that does not inhibit binding of thecomplex of the Tim protein bound to the carrier and the extracellularmembrane vesicle or the virus in the sample. Examples of the surfactantinclude Tween 20, or the like, and concentration of the surfactant inthe solution, for containing the extracellular membrane vesicles or theviruses pertaining to the present invention, is usually 0.00001 to 0.2%,and preferably it is usually 0.0005 to 0.1%.

<Calcium Ion Concentration and Origin of Calcium Ion>

In the present invention, the calcium ion is present in formation of thecomplex of the Tim protein pertaining to the present invention, thecarrier, and the extracellular membrane vesicle or the virus in thesample. More specifically, the calcium ion is present in bringing intocontacted the Tim protein pertaining to the present invention, and theextracellular membrane vesicle or the virus in the sample.

Calcium ion concentration, in bringing into contacted the Tim proteinpertaining to the present invention, and the extracellular membranevesicle or the virus in the sample pertaining to the present invention,is usually 0.5 to 100 mM, preferably 1.0 to 10 mM, and more preferably2.0 to 5.0 mM. It should be noted that it is not without saying that thecalcium ion of the concentration as above is necessary, in the solutioncontaining the complex, until the complex of the Tim protein pertainingto the present invention, and the extracellular membrane vesicle or thevirus in the sample pertaining to the present invention is formed, andup to carrying out the obtaining step, that is, until the complex issubjected to the separation step.

In addition, source of the calcium ion is not especially limited, andincludes, for example, calcium chloride, calcium hydroxide, calciumhydrogen carbonate, calcium iodide, calcium bromide, calcium acetate, orthe like; preferably calcium chloride, calcium hydrogen carbonate, andcalcium iodide; and more preferably calcium chloride, and calciumhydrogen carbonate.

It should be noted that, as the method for the calcium ion to bepresent, when the Tim protein pertaining to the present invention, andthe extracellular membrane vesicle or the virus in the sample is broughtinto contacted, generally, the calcium ion as described above may becontained in the solution containing the Tim carrier of the presentinvention, the solution containing the sample and the solutioncontaining the Tim protein pertaining to the present invention, and/orthe solution containing the carrier pertaining to the present invention,in such an amount that attains calcium ion concentration in a rangedescribed above, when the Tim protein pertaining to the presentinvention, the carrier pertaining to the present invention, and theextracellular membrane vesicle or the virus in the sample is broughtinto contacted. In addition, using a solution containing the calciumion, in such an amount that attains calcium ion concentration in a rangedescribed above, when the Tim protein pertaining to the presentinvention, the carrier pertaining to the present invention, and theextracellular membrane vesicle or the virus in the sample is broughtinto contacted, this solution, the solution containing the Tim proteinof the present invention, the solution containing the sample and thesolution containing the Tim protein pertaining to the present invention,and/or the carrier pertaining to the present invention may be mixed.

The solution for containing the calcium ion is the same as the solutionfor containing (dissolving or suspending) the extracellular membranevesicle or the virus pertaining to the present invention, and specificexamples, and the like, are also the same.

<Amount of Sample>

Amount of the sample to be brought into contacted with 1 μg of the Timprotein of the present invention, in the complex formation step, isusually 0.1 to 100 mL, preferably 0.1 to 10 mL, and more preferably 0.1to 1.0 mL.

<Temperature>

Temperature in bringing into contacted the Tim protein of the presentinvention, with the extracellular membrane vesicles or the viruses inthe sample, in the complex formation step, is usually 4° to 37° C.,preferably 4 to 25° C., and more preferably 4 to 11° C.

<Time>

Contacting time of the Tim protein pertaining to the present inventionand the sample, in the complex formation step, is usually 0.5 to 24hours, preferably 0.5 to 8 hours, and more preferably 0.5 to 4 hours.

The complex formation step is classified into two cases of (1-A) wherethe Tim carrier of the present invention, prepared in advance, is used(that is, the case where the carrier on which the Tim protein pertainingto the present invention is bound is used), and (1-B) where the Timprotein pertaining to the present invention and the carrier pertainingto the present invention are used separately.

<(1-A)>

(1-A) is a method for forming the complex of the Tim protein bound tothe carrier pertaining to the present invention, and the extracellularmembrane vesicle or the virus in the sample (hereafter it may beabbreviated as “the complex pertaining to the present invention”, insome cases), by bringing into contacted the Tim carrier of the presentinvention, and the extracellular membrane vesicle or the virus in thesample, in the presence of the calcium ion. In (1-A), the calcium ionmay be present, when the Tim carrier of the present invention and theextracellular membrane vesicle or the virus in the sample is broughtinto contacted. Specifically, the solution containing the carrier onwhich the Tim protein pertaining to the present invention is bound andcontaining the calcium ion and/or the solution containing the sample andthe calcium ion may be used, or the solution containing the carrier onwhich the Tim protein pertaining to the present invention is bound, andthe solution containing the sample and the calcium ion may be used.

—Amount of Sample Pertaining to the Present Invention—

Amount of the sample to be brought into contacted with 1 mg of the Timcarrier of the present invention, in (1-A), is usually 0.1 to 100 mL,preferably 0.1 to 10 mL, and more preferably 0.1 to 1.0 mL.

—Contacting Temperature—

Temperature when the Tim carrier of the present invention, and thesample are brought into contacted, in (1-A), is usually 4.0 to 37° C.,preferably 4.0 to 25° C., and more preferably 4.0 to 11° C.

—Contacting Time—

Contacting time of the Tim carrier of the present invention, and thesample (the extracellular membrane vesicles or the viruses), in (1-A),is usually 0.5 to 24 hours, preferably 0.5 to 8.0 hours, and morepreferably 0.5 to 4.0 hours.

—Amount of Tim Carrier of the Present Invention—

Amount of the Tim carrier of the present invention, in (1-A), is usually0.1 to 20 mg, preferably 0.3 to 10 mg, and more preferably 0.5 to 6.0mg, relative to 1 mL of the solution in forming the complex pertainingto the present invention.

Specific Example of 1-A

(1-A) may be carried out, for example, by the following method. That is,the complex of the Tim protein bound to the carrier pertaining to thepresent invention, and the extracellular membrane vesicles or theviruses in the sample is formed by bringing into contacted usually 0.1to 20 mg, preferably 0.3 to 10 mg, and more preferably 0.5 to 6.0 mg ofthe Tim carrier of the present invention, relative to 1 mL of thesolution after mixing of the Tim carrier of the present invention, andthe solution containing the sample and the solution containing thecalcium ion (the solution in forming the complex pertaining to thepresent invention), with the solution containing the calcium ion, in anamount to attain a calcium ion concentration of usually 0.5 to 100 mM,preferably 1.0 to 10 mM, and more preferably 2.0 to 5.0 mM, in thesolution in forming the complex pertaining to the present invention, andusually 0.1 to 100 mL, preferably 0.1 to 10 mL, and more preferably 0.1to 1.0 mL of the sample, relative to 1 mg of the Tim carrier of thepresent invention, at usually 4.0 to 37° C., preferably 4.0 to 25° C.,and more preferably 4.0 to 11° C., for usually 0.5 to 24 hours,preferably 0.5 to 8.0 hours, and more preferably 0.5 to 4.0 hours.

<(1-B)>

(1-B) is a method for using the Tim protein of the present invention,and the carrier pertaining to the present invention separately. Forexample, when the Tim protein pertaining to the present invention andthe carrier pertaining to the present invention are bound, utilizingaffinity binding, it may be carried out as the following (1-B-i),(1-B-ii), or (1-B-iii).

(1-B-i)

The complex pertaining to the present invention is formed by bringinginto contacted the Tim protein of the present invention, the carrierpertaining to the present invention, and the extracellular membranevesicle or the virus in the sample, at the same time, in the presence ofthe calcium ion.

(1-B-ii)

The complex pertaining to the present invention is formed by forming thecomplex of the Tim protein of the present invention, and theextracellular membrane vesicle or the virus by bringing into contactedthe Tim protein of the present invention, and the extracellular membranevesicle or the virus in the sample, in the presence of the calcium ion,and then by further bringing into contacted the complex and the carrierpertaining to the present invention.

(1-B-iii)

The complex pertaining to the present invention is formed by bringinginto contacted the carrier of the present invention, and theextracellular membrane vesicle or the virus in the sample, and then byfurther bringing into contacted the Tim protein of the presentinvention, in the presence of the calcium ion.

—(1-B-i)—

(1-B-i) is a method for forming the complex pertaining to the presentinvention composed of [the extracellular membrane vesicle or thevirus-(the Tim protein pertaining to the present invention)-the affinitysubstance-(the carrier pertaining to the present invention)], bybringing into contacted the Tim protein pertaining to the presentinvention, the carrier pertaining to the present invention, and theextracellular membrane vesicle or the virus in the sample, at the sametime, in the presence of the calcium ion.

Specifically, for example, in using two or more kinds of substanceshaving affinity each other, as the affinity substance, the complexpertaining to the present invention composed of [the extracellularmembrane vesicle or the virus in the sample-(the Tim protein pertainingto the present invention)-the affinity substance-(the carrier pertainingto the present invention)] is formed by bringing into contacted the Timprotein pertaining to the present invention bound with either of theaffinity substances (the affinity substance-binding Tim protein), andthe carrier bound with the remaining affinity substances (the affinitysubstance-binding carrier), and the extracellular membrane vesicle orthe virus in the sample, at the same time, in the presence of thecalcium ion, to bind the Tim protein pertaining to the present inventionin the affinity substance-binding Tim protein, and the extracellularmembrane vesicle or the virus, as well as to bind the affinity substancein the affinity substance-binding Tim protein, and the affinitysubstance in the affinity substance-binding carrier.

In (1-B-i), the calcium ion may be present in bringing into contactedthe Tim protein pertaining to the present invention, the carrierpertaining to the present invention, and the extracellular membranevesicle or the virus in the sample, at the same time. Specifically, itmay be carried out either by using the solution containing the Timprotein pertaining to the present invention, the solution containing thecarrier pertaining to the present invention, and the sample, which thecalcium ion contained in at least one more kinds of the solutions, or bybringing into contacted the solution containing the Tim proteinpertaining to the present invention, the solution containing the carrierpertaining to the present invention, and the solution containing thesample and the calcium ion.

—Amount of Sample Pertaining to the Present Invention—

In (1-B-i), an amount of the sample to be brought into contacted with 1μg of the Tim protein pertaining to the present invention is usually 0.1to 100 mL, preferably 0.1 to 10 mL, and more preferably 0.1 to 1.0 mL.

—Amount of Tim Protein Pertaining to the Present Invention—

In (1-B-i), an amount of the Tim protein pertaining to the presentinvention is usually 0.01 to 200 μg, preferably 0.15 to 50 μg, and morepreferably 0.5 to 24 μg, relative to 1 mL of the solution in forming thecomplex pertaining to the present invention.

—Amount of Carrier Pertaining to the Present Invention—

In (1-B-i), an amount of the carrier pertaining to the present inventionis usually 1 to 20 mg, preferably 0.3 to 10 mg, and more preferably 0.5to 6.0 mg, relative to 1 mL of the solution in forming the complexpertaining to the present invention.

—Contacting Temperature—

In (1-B-i), temperature in bringing into contacted the Tim protein ofthe present invention, the carrier pertaining to the present invention,and the extracellular membrane vesicle or the virus in the sample, isusually 4 to 37° C., preferably 4 to 25° C., and more preferably 4 to11° C.

—Contacting Time—

Contacting time in bringing into contacted the Tim protein of thepresent invention, the carrier pertaining to the present invention, andthe extracellular membrane vesicle or the virus in the sample, isusually 0.5 to 24 hours, preferably 0.5 to 8.0 hours, and morepreferably 0.5 to 4.0 hours.

It should be noted that contact of the Tim protein of the presentinvention, the carrier pertaining to the present invention, and theextracellular membrane vesicle or the virus in the sample is carried outusually by bringing into contacted the solution containing the Timprotein of the present invention, the carrier pertaining to the presentinvention, the extracellular membrane vesicle or the virus in thesample, and the solution containing the calcium ion.

The solution for containing the Tim protein pertaining to the presentinvention may be any solution which is capable of dissolving the Timprotein pertaining to the present invention in a stable state, and doesnot inhibit binding of the Tim protein pertaining to the presentinvention, the carrier pertaining to the present invention and theextracellular membrane vesicle or the virus, and includes for example,purified water, or a buffer solution which has buffer action, forexample, at pH 7.0 to 8.0, and preferably 7.2 to 7.6 (for example, PBS,TBS, HBS or the like). In addition, buffer agent concentration in thesebuffer solutions is appropriately selected from a range of usually 5 to50 mM, and preferably 10 to 30 mM, and NaCl concentration isappropriately selected from a range of usually 100 to 200 mM, andpreferably 140 to 160 mM. For example, saccharides, salts, such as NaCl,and the like, a surfactant, a preservative, a protein, or the like, maybe contained in this solution, as long as in such an amount thatdissolves the Tim protein pertaining to the present invention in astable state, and does not inhibit binding of the Tim protein pertainingto the present invention, the carrier pertaining to the presentinvention and the extracellular membrane vesicle or the virus. Thesurfactant includes, for example, Tween 20, or the like, and surfactantconcentration in the solution for containing the Tim protein pertainingto the present invention is usually 0.00001 to 0.2%, and preferably0.0005 to 0.1%.

The solution for containing the calcium ion is the same as the solutionfor containing (dissolving or suspending) the extracellular membranevesicle or the virus pertaining to the present invention, and thespecific example, or the like, is also the same.

The solution containing the calcium ion is the solution containing suchamount of the calcium ion that attains a calcium ion concentration ofusually 0.5 to 100 mM, preferably 1 to 10 mM, and more preferably 2 to 5mM in the solution in forming the complex pertaining to the presentinvention, among the solutions containing the calcium ion.

Specific Example of (1-B-i)

(1-B-i) may be carried out, for example, by the following method. Thatis, the complex pertaining to the present invention is formed bybringing into contacted usually 0.5 μL to 1 ml, preferably 0.5 μL to 100μL, and more preferably 0.5 μL to 10 μL of the solution containingusually 0.01 to 200 μg, preferably 0.15 to 50 μg, and more preferably0.5 to 24 μg of the Tim protein pertaining to the present invention (forexample, the solution containing the Tim protein of the presentinvention, for example, in purified water or, in the buffer solutionwhich has buffer action at pH 7.0 to 8.0, and preferably 7.2 to 7.6),relative to 1 mL of the solution in forming the complex pertaining tothe present invention; usually 0.1 to 100 ml, preferably 0.1 to 10 ml,and more preferably 0.1 to 1 ml of the sample, relative to 1 μg of theTim protein pertaining to the present invention; the solution containingthe calcium ion in such an amount that attains a concentration of thecalcium ion of usually 0.5 to 100 mM, preferably 1 to 10 mM, and morepreferably 2 to 5 mM, in the solution in forming the complex pertainingto the present invention; and usually 0.1 to 20 mg, preferably 0.3 to 10mg, and more preferably 0.5 to 6 mg of the carrier pertaining to thepresent invention, relative to 1 mL of a solution after mixing thesolution containing the Tim protein pertaining to the present invention,the solution containing the sample and the calcium ion, and the solutioncontaining the carrier pertaining to the present invention (the solutionin forming the complex pertaining to the present invention), for usually0.5 to 24 hours, preferably 0.5 to 8 hours, and more preferably 0.5 to 4hours.

—(1-B-ii)—

(1-B-ii) is a method for forming the complex of the Tim protein of thepresent invention, and the extracellular membrane vesicle or the virus,by bringing into contacted the Tim protein of the present invention, andthe extracellular membrane vesicle or the virus in the sample, in thepresence of the calcium ion, and further by bringing into contacted thecomplex and the carrier pertaining to the present invention to form thecomplex composed of [the extracellular membrane vesicle or thevirus-(the Tim protein pertaining to the present invention)-the affinitysubstance-(the carrier pertaining to the present invention)].

Specifically, for example, in using two or more kinds of substanceshaving affinity each other, as the affinity substances, the complex of[the extracellular membrane vesicle or the virus-(the Tim proteinpertaining to the present invention)-the affinity substance] is formedby bringing into contacted the Tim protein bound with either of theaffinity substances (the affinity substance-binding Tim protein), andthe extracellular membrane vesicle or the virus in the sample, in thepresence of the calcium ion to bind the Tim protein in the affinitysubstance-binding Tim protein, and the extracellular membrane vesicle.Next, [the extracellular membrane vesicle or the virus-(the Tim proteinpertaining to the present invention-the affinity substance)-(theaffinity substance-the carrier pertaining to the present invention)] isformed by bringing into contacted the [the extracellular membranevesicle-(the Tim protein pertaining to the present invention-theaffinity substance)] complex, and the carrier bound with the otheraffinity substance (the affinity substance-binding carrier) to bind theaffinity substance in [the extracellular membrane vesicle or thevirus-(the Tim protein pertaining to the present invention-the affinitysubstance)] complex, and the affinity substance in the affinitysubstance-binding carrier.

In addition, for example, in using a substance having affinity to anaffinity tag or an anti-Tim antibody, as the affinity substance, [theextracellular membrane vesicle or the virus-the Tim protein pertainingto the present invention] complex is formed, by bringing into contactedthe Tim protein pertaining to the present invention and theextracellular membrane vesicle or the virus in the sample, in thepresence of the calcium ion to bind the Tim protein pertaining to thepresent invention and the extracellular membrane vesicle or the virus.Next, [the extracellular membrane vesicle or the virus-the Tim proteinpertaining to the present invention-(the affinity substance-the carrierpertaining to the present invention)] complex is formed, by bringinginto contacted [the extracellular membrane vesicle or the virus-the Timprotein pertaining to the present invention] complex, and the carrierbound with the affinity substance (the affinity substance-bindingcarrier) to bind the Tim protein pertaining to the present invention in[the extracellular membrane vesicle or the virus-the Tim proteinpertaining to the present invention] complex, and the affinity substancein the affinity substance-binding carrier.

In (1-B-ii), the calcium ion may be present in bringing into contactedthe Tim protein pertaining to the present invention, and theextracellular membrane vesicle or the virus in the sample. Specifically,the solution containing the Tim protein pertaining to the presentinvention and/or the sample, which is containing the calcium ion may beused, or the solution containing the Tim protein pertaining to thepresent invention, the sample, and the solution containing the calciumion may be used.

—Amount of Sample Pertaining to the Present Invention—

An amount of the sample to be brought into contacted with 1 μg of theTim protein pertaining to the present invention in (1-B-ii), is usually0.1 to 100 ml, preferably 0.1 to 10 ml, and more preferably 0.1 to 1.0ml.

—Amount of Tim Protein Pertaining to the Present Invention—

An amount of the Tim protein pertaining to the present invention in(1-B-ii) is usually 0.01 to 200 μg, preferably 0.15 to 50 μg, and morepreferably 0.5 to 24 μg, relative to 1 mL of the solution in forming thecomplex pertaining to the present invention.

—Amount of Carrier Pertaining to the Present Invention—

An amount of the carrier pertaining to the present invention in (1-B-ii)is usually 0.1 to 20 mg, preferably 0.3 to 10 mg, and more preferably0.5 to 6 mg, relative to 1 mL of the solution in forming the complexpertaining to the present invention.

—Contacting Temperature—

Temperature in bringing into contacted the Tim protein of the presentinvention, and the extracellular membrane vesicles or the viruses in thesample in (1-B-ii), is usually 4 to 37° C., preferably 4 to 25° C., andmore preferably 4 to 11° C.

After forming the complex of the Tim protein of the present inventionand extracellular membrane vesicle or the virus in (1-B-ii), temperaturein further bringing into contacted the complex and the carrierpertaining to the present invention is usually 4 to 37° C., preferably 4to 25° C., and more preferably 4 to 11° C.

—Contacting Time—

Contacting time of the Tim protein of the present invention, the carrierpertaining to the present invention, and the extracellular membranevesicle or the virus in the sample pertaining to present invention in(1-B-ii), is usually 0.5 to 24 hours, preferably 0.5 to 8 hours, andmore preferably 0.5 to 4 hours.

After forming the complex of the Tim protein of the present inventionand extracellular membrane vesicle or the virus in (1-B-ii), time forfurther bringing into contacted the complex and the carrier pertainingto the present invention is usually 0.5 to 24 hours, preferably 0.5 to 8hours, and more preferably 0.5 to 4 hours.

It should be noted that contact of the Tim protein pertaining to thepresent invention and the extracellular membrane vesicle or the virus inthe sample is usually carried out by bringing into contacted thesolution containing the Tim protein pertaining to the present invention,the sample and the solution containing the calcium ion.

The solution for containing the Tim protein pertaining to the presentinvention may be any solution, as long as it is capable of dissolvingthe Tim protein pertaining to the present invention in a stable state,and dose not inhibit binding of the Tim protein pertaining to thepresent invention, the carrier pertaining to the present invention andthe extracellular membrane vesicle or the virus pertaining to thepresent invention, and includes for example, purified water, or a buffersolution which has buffer action, for example, at pH 7.0 to 8.0, andpreferably 7.2 to 7.6 (for example, TBS, HBS or the like). In addition,buffer agent concentration in these buffer solutions is appropriatelyselected from a range of usually 5 to 50 mM, and preferably 10 to 30 mM,and NaCl concentration is appropriately selected from a range of usually100 to 200 mM, and preferably 140 to 160 mM. For example, saccharides,salts, such as NaCl, and the like, a surfactant, a preservative, aprotein, or the like, may be contained in this solution, as long as insuch an amount that dissolves the Tim protein pertaining to the presentinvention in a stable state, and dose not inhibit binding of the Timprotein pertaining to the present invention, the carrier pertaining tothe present invention, and the extracellular membrane vesicle or thevirus. The surfactant includes, for example, Tween 20, or the like, andsurfactant concentration in the solution for containing the Tim proteinpertaining to the present invention is usually 0.00001 to 0.2%, andpreferably 0.0005 to 0.1%.

The solution for containing the calcium ion is the same as the solutionfor containing (dissolving or suspending) the extracellular membranevesicle or the virus pertaining to the present invention. The specificexample, or the like, is also the same.

The solution containing the calcium ion is the solution containing suchan amount of the calcium ion that attains a calcium ion concentration ofusually 0.5 to 100 mM, preferably 1 to 10 mM, and more preferably 2 to 5mM, in the solution in forming the complex pertaining to the presentinvention.

Specific Example of (1-B-ii)

(1-B-ii) may be carried out, for example, by the following method. Thatis, the complex of the Tim protein of the present invention and theextracellular membrane vesicles or the viruses is formed, by bringinginto contacted usually 0.5 μL to 1 mL, preferably 0.5 μL to 100 μL, andmore preferably 0.5 μL to 10 μL of the solution containing usually 0.01to 200 μg, preferably 0.15 to 50 μg, and more preferably 0.5 to 24 μg ofthe Tim protein pertaining to the present invention (the solutioncontaining the Tim protein pertaining to the present invention, forexample, in purified water, or in the buffer solution, having bufferingaction, for example, at pH 7.0 to 8.0, and preferably 7.2 to 7.6),relative to 1 mL of the solution in forming the complex pertaining tothe present invention; usually 0.1 to 100 mL, preferably 0.1 to 10 mL,and more preferably 0.1 to 1 mL of the sample, relative to 1 μg of theTim protein pertaining to the present invention; and the solutioncontaining the calcium ion, in such an amount that attains a calcium ionconcentration of usually 0.5 to 100 mM, preferably 1 to 10 mM, and morepreferably 2 to 5 mM, in a solution mixed with the sample, the solutioncontaining the calcium ion, and the solution containing the Tim proteinpertaining to the present invention, and further in a solution afterbringing into contacted the mixed solution and the carrier pertaining tothe present invention; at usually 4 to 37° C., preferably 4 to 25° C.,and more preferably 4 to 11° C., for usually 0.5 to 24 hours, preferably0.5 to 8 hours, and more preferably 0.5 to 4 hours, and after that, thecomplex pertaining to the present invention is formed, by the furtheraddition of usually 0.1 to 20 mg, preferably 0.3 to 10 mg, and morepreferably 0.5 to 6 mg of the carrier pertaining to the presentinvention, relative to 1 mL of the solution in formation of the complexpertaining to the present invention, and by bringing into contacted theresulting complex of the Tim protein of the present invention and theextracellular membrane vesicles or the viruses, and the carrierpertaining to the present invention, at usually 4 to 37° C., preferably4 to 25° C., and more preferably 4 to 11° C., for usually 0.5 to 24hours, preferably 0.5 to 8 hours, and more preferably 0.5 to 4 hours.

—(1-B-iii)—

(1-B-iii) is a method for forming the complex pertaining to the presentinvention composed of [the extracellular membrane vesicle or thevirus-(the Tim protein pertaining to the present invention)-the affinitysubstance-(the carrier pertaining to the present invention)], bybringing into contacted the carrier of the present invention, and theextracellular membrane vesicle or the virus in the sample, and afterthat by further bringing into contacted the Tim protein of the presentinvention, in the presence of the calcium ion.

Specifically, for example, in using two or more kinds of substanceshaving affinity each other as the affinity substance, [the extracellularmembrane vesicle or the virus-(the Tim protein pertaining to the presentinvention-the affinity substance)-(the affinity substance-the carrierpertaining to the present invention)] complex is formed by bringing intocontacted the carrier bound with either of the affinity substances (theaffinity substance-binding carrier), and the extracellular membranevesicle or the virus in the sample, and next by bringing into contactedwith the Tim protein bound with the other affinity substance (theaffinity substance-binding Tim protein), at the same time, in thepresence of the calcium ion to bind the Tim protein in the affinitysubstance-binding Tim protein, and the extracellular membrane vesicle orthe virus, as well as to bind the affinity substance in the affinitysubstance-binding Tim protein, and the affinity substance in theaffinity substance-binding carrier.

In addition, in using, for example, a substance having affinity to theaffinity tag or an anti-Tim antibody, as the affinity substance, [theextracellular membrane vesicle or the virus-the Tim protein pertainingto the present invention-(the affinity substance-the carrier pertainingto the present invention)] complex is formed, by bringing into contactedthe carrier bound with the affinity substance (the affinitysubstance-binding carrier), and the extracellular membrane vesicle orthe virus in the sample, and next by bringing into contacted with theTim protein pertaining to the present invention, at the same time, inthe presence of the calcium ion to bind the Tim protein pertaining tothe present invention and the extracellular membrane vesicle or thevirus, as well as to bind the Tim protein pertaining to the presentinvention, and the affinity substance in the affinity substance-bindingcarrier.

In (1-B-iii), the calcium ion may be present in bringing into contactedthe Tim protein pertaining to the present invention, and theextracellular membrane vesicle or the virus in the sample. Specifically,either the solution containing the Tim protein pertaining to the presentinvention, and/or the solution containing the calcium ion in theextracellular membrane vesicle or the virus in the sample may be used,or the solution containing the Tim-4 protein pertaining to the presentinvention, and the solution containing the extracellular membranevesicle or the virus in the sample and the calcium ion may be used.

—Amount of Sample Pertaining to the Present Invention—

An amount of the sample to be brought into contacted with 1 μg of theTim protein pertaining to the present invention in (1-B-iii), is usually0.1 to 100 ml, preferably 0.1 to 10 ml, and more preferably 0.1 to 1 ml.

—Amount of Tim Protein Pertaining to the Present Invention—

An amount of the Tim protein pertaining to the present invention in(1-B-iii) is usually 0.01 to 200 μg, preferably 0.15 to 50 μg, and morepreferably 0.5 to 24 μg, relative to 1 mL of the solution in forming thecomplex pertaining to the present invention.

—Amount of Carrier Pertaining to the Present Invention—

An amount of the carrier pertaining to the present invention in(1-B-iii) is usually 0.1 to 20 mg, preferably 0.3 to 10 mg, and morepreferably 0.5 to 6 mg, relative to 1 mL of the solution in forming thecomplex pertaining to the present invention.

—Contacting Temperature—

Temperature in bringing into contacted the carrier pertaining to thepresent invention and the extracellular membrane vesicle or the virus inthe sample in (1-B-iii), is usually 4 to 37° C., preferably 4 to 25° C.,and more preferably 4 to 11° C.

Temperature in further bringing into contacted the Tim protein of thepresent invention, after bringing into contacted the carrier of thepresent invention, and the extracellular membrane vesicle or the virusin the sample, in (1-B-iii), is usually 4 to 37° C., preferably 4 to 25°C., and more preferably 4 to 11° C.

—Contacting Time—

Contacting time in bringing into contacted the carrier pertaining to thepresent invention and the sample in (1-B-iii), is usually 0.5 to 24hours, preferably 0.5 to 8 hours, and more preferably 0.5 to 4 hours.

Contacting time in further bringing into contacted the Tim proteinpertaining to the present invention, after bringing into contacted thecarrier of the present invention and the extracellular membrane vesicleor the virus in the sample in (1-B-iii), is usually 0.5 to 24 hours,preferably 0.5 to 8 hours, and more preferably 0.5 to 4 hours.

It should be noted that further contact with the Tim protein pertainingto the present invention, after bringing into contacted the carrier ofthe present invention, and the extracellular membrane vesicle or thevirus in the sample, is generally carried out by using the solutioncontaining the Tim protein pertaining to the present invention, and thesolution containing the calcium ion.

The solution for containing the Tim protein pertaining to the presentinvention may be any solution, as long as it is capable of dissolvingthe Tim protein pertaining to the present invention in a stable state,and dose not inhibit binding of the Tim protein pertaining to thepresent invention, the carrier pertaining to the present invention andthe extracellular membrane vesicle or the virus, and includes forexample, purified water, or the buffer solution which has buffer action,for example, at pH 7.0 to 8.0, and preferably 7.2 to 7.6 (for example,PBS, TBS, HBS or the like). In addition, buffer agent concentration inthese buffer solutions is appropriately selected from a range of usually5 to 50 mM, and preferably 10 to 30 mM, and NaCl concentration isappropriately selected from a range of usually 100 to 200 mM, andpreferably 140 to 160 mM. For example, saccharides, salts, such as NaCl,and the like, a surfactant, a preservative, a protein, or the like, maybe contained in this solution, as long as in such an amount thatdissolves the Tim-4 protein pertaining to the present invention in astable state, and dose not inhibit binding of the Tim protein pertainingto the present invention, the carrier pertaining to the presentinvention and the extracellular membrane vesicle or the virus. Thesurfactant includes, for example, Tween 20, or the like, and surfactantconcentration in the solution for containing the Tim protein pertainingto the present invention is usually 0.00001 to 0.2%, and preferably0.0005 to 0.1%.

The solution for containing the calcium ion is the same as the solutionfor containing (dissolving or suspending) the extracellular membranevesicle or the virus pertaining to the present invention, and thespecific examples are also the same.

The solution containing the calcium ion is the solution having such anamount of the calcium ion that attains a calcium ion concentration ofusually 0.5 to 100 mM, preferably 1 to 10 mM, and more preferably 2 to 5mM, in the solution in forming the complex pertaining to the presentinvention.

Specific Example of (1-B-iii)

(1-B-iii) may be carried out, for example, by the following method. Thatis, the complex pertaining to the present invention is formed bybringing into contacted usually 0.1 to 100 ml, preferably 0.1 to 10 ml,and more preferably 0.1 to 1 ml of the sample pertaining to the presentinvention, relative to 1 μg of the Tim protein pertaining to the presentinvention, and usually 0.1 to 20 mg, preferably 0.3 to 10 mg, and morepreferably 0.5 to 6 mg of the carrier pertaining to the presentinvention, relative to 1 mL of the solution in forming the complexpertaining to the present invention, at usually 4 to 37° C., preferably4 to 25° C., and more preferably 4 to 11° C., for usually 0.5 to 24hours, preferably 0.5 to 8 hours, and more preferably 0.5 to 4 hours,and after that, by the addition of the solution containing such amountof the calcium ion that attains a calcium ion concentration of usually0.5 to 100 mM, preferably 1.0 to 10 mM, and more preferably 2 to 5 mM,in the solution obtained by bringing into contacted the solutioncontaining the sample pertaining to the present invention and thecalcium ion, and the carrier pertaining to the present invention, andfurther by bringing into contacted the solution containing the Timprotein pertaining to the present invention; and usually 0.5 μL to 1 mL,preferably 0.5 μL to 100 μL, and more preferably 0.5 μL to 10 μL of thesolution containing usually 0.01 to 200 μg, preferably 0.15 to 50 μg,and more preferably 0.5 to 24 μg of the Tim protein pertaining to thepresent invention (the solution containing the Tim protein pertaining tothe present invention, for example, in purified water or in the buffersolution, which has buffer action, for example, at pH 7.0 to 8.0, andpreferably 7.2 to 7.6), relative to 1 mL of the solution in forming thecomplex pertaining to the present invention, to bring into contacted atusually 4 to 37° C., preferably 4 to 25° C., and more preferably 4 to11° C., for usually 0.5 to 24 hours, preferably 0.5 to 8 hours, and morepreferably 0.5 to 4 hours.

<6-2. Complex Separation Step>

The complex separation step in the obtaining method of the presentinvention is an obtaining step of the separated complex pertaining tothe present invention, after the complex formation step, by separationof the complex of the Tim protein bound to the resulting carrierpertaining to the present invention (the Tim carrier of the presentinvention), and the extracellular membrane vesicle or the virus in thesample (the complex pertaining to the present invention), and thesample.

The complex separation step in the obtaining method of the presentinvention may be any method, as long as it is capable of obtaining thecomplex pertaining to the present invention, by separation of thecomplex pertaining to the present invention, and the sample, andincludes, for example, the following methods.

(1) When a magnetic carrier is used as the carrier pertaining to thepresent invention, a method for installing a container, used in thecomplex formation step, in the magnet stand as needed, collecting thecomplex pertaining to the present invention on the tube wall by usingmagnetic force and removing the supernatant sample to separate them.(2) When the carrier of the present invention is beads-like, a methodfor subjecting the container, used in the complex formation step, tocentrifugal separation treatment to collect the complex pertaining tothe present invention as precipitate, and to remove the supernatantsample to separate them.(3) When the carrier of the present invention in a form of anon-beads-like plate, or the like, is used, a method for separatingthese by removing only the sample.(4) A method for separating the complex pertaining to the presentinvention, and the sample by filtration.

After separation of the complex of the present invention and the sample,in this way, the separated complex of the present invention may beobtained (recovered) by a method known per se.

Specific Example of Complex Separation Step

When the magnetic carrier is used as the carrier pertaining to thepresent invention, the container used in the complex formation step isinstalled in the magnet stand, as needed, and the complex pertaining tothe present invention is collected on the tube wall by using magneticforce to remove the supernatant sample.

<6-3. Washing Operation>

After the complex formation step and the complex separation step, theresulting complex pertaining to the present invention may be washed, asneeded, using the calcium ion-containing washing solution (hereinafter,it may be optionally abbreviated as “washing operation”). Impurities inthe sample pertaining to the present invention, such as componentsoriginated from the cell attached on the surface of the carrierpertaining to the present invention, and the like, can be removed by thewashing operation. A washing method usually practiced in this field canbe used, except for using the washing solution containing the calciumion, as described above, as the washing method. The calciumion-containing washing solution, to be used in the washing operation,may be any solution, as long as it contains usually 0.5 to 100 mM,preferably 1 to 10 mM, and more preferably 2 to 5 mM of the calcium ion,and does not influence binding of the extracellular membrane vesicle orthe virus pertaining to the present invention in the complex, the Tim-4protein pertaining to the present invention, and the carrier of thepresent invention, and includes, for example, the buffer solutioncontaining usually 0.5 to 100 mM, preferably 1 to 10 mM, and morepreferably 2 to 5 mM of the calcium ion, and having buffer action at pH7.0 to 8.0, and preferably 7.2 to 7.6, and not precipitating calcium(for example, TBS, HBS). It should be noted that the phosphate buffer isnot preferable, because it precipitates by binding with calcium. Inaddition, buffer agent concentration in these buffer solutions isselected from a range of usually 5 to 50 mM, and preferably 10 to 30 mM,and NaCl concentration is selected from a range of usually 100 to 200mM, and preferably 140 to 160 mM. This solution may contain, forexample, saccharides, salts, such as NaCl, and the like, a surfactant, apreservative, a protein, or the like, as long as in such an amount thatdoes not inhibit binding of the extracellular membrane vesicle or thevirus pertaining to the present invention in the complex, the Timprotein pertaining to the present invention, and the carrier of thepresent invention. The surfactant includes, for example, Tween 20(produced by Wako Pure Chemical Industries Co.), or the like, andsurfactant concentration in the washing solution is usually 0.00001 to0.2%, and preferably 0.0005 to 0.1%.

Explanation will be given on an example by taking the case of using themagnetic particles, as the carrier pertaining to the present invention.First of all, the calcium ion-containing washing solution is added inthe container containing the resulting complex pertaining to the presentinvention by the complex separation step, and is stirred. After that,the container is installed in the magnet stand, to collect the complexon the tube wall by using magnetic force, and the solution in thecontainer is discarded. The washing operation may be repeated severaltimes, as needed.

Specific Example of Washing Operation

First of all, the calcium ion-containing washing solution (for example,the buffer solution containing usually 0.5 to 100 mM, preferably 1 to 10mM, and more preferably 2 to 5 mM of the calcium ion, having bufferaction at pH 7.0 to 8.0, and preferably 7.2 to 7.6, and notprecipitation calcium. However, the phosphate buffer is not preferablebecause it precipitates by binding with calcium) is added into thecontainer containing the resulting complex pertaining to the presentinvention by the complex separation step, and is stirred. After that,the container is installed in the magnet stand, to collect the complexon the tube wall by using magnetic force, and solution in the containeris discarded. The washing operation may be repeated several times, asneeded.

<6-4. Obtaining Step>

The obtaining step is a step for obtaining the extracellular membranevesicle or the virus pertaining to the present invention, after carryingout the complex formation step, the complex separation step, andcarrying out the washing step (washing operation), as needed, toseparate the extracellular membrane vesicle or the virus from thecomplex of the Tim protein bound to the resulting carrier pertaining tothe present invention, and the extracellular membrane vesicle or thevirus in the sample.

In this way, the extracellular membrane vesicle or the virus pertainingto the present invention can be obtained in high purity.

Specific examples of the obtaining step include, for example, thefollowing

(2-A) and (2-B).

(2-A) A method for using a protein denaturing agent.

(2-B) A method for decreasing calcium ion concentration.

<(2-A): Method for Using Protein Denaturing Agent>

(2-A) is a step for separating the extracellular membrane vesicle or thevirus from the complex pertaining to the present invention, aftercarrying out the complex formation step, the complex separation step,and carrying out the washing operation, as needed, to make acting theprotein denaturing agent on the resulting complex pertaining to thepresent invention, to denature the Tim protein pertaining to the presentinvention in the complex pertaining to the present invention. In thisway, the extracellular membrane vesicle or the virus pertaining to thepresent invention can be obtained in high purity.

—Protein Denaturing Agent—

The protein denaturing agent to be used in (2-A) may be any one, as longas it is generally used in this field, as a compound to denature theprotein, and includes an anionic surfactant, for example, SDS (sodiumdodecyl sulfate),

N-lauroyl sarcosine, or the like; an amphoteric surfactant, such asCHAPS (3-(3-cholamidepropyl)dimethylamino-1-propane sulfonate salt),Zwittergent 3-12 (N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate),or the like; a non-ionic surfactant, such as Brij 35 (produced by TakaraBio Inc.), dodecyl-β-D-maltoside(n-dodecyl-β-D-maltoside), Nonidet P-40,octyl-β-D-glucoside (octyl-β-D-glcoside), Triton X-100 (polyoxyethylene(10) octylphenol), Tween 20 (polyoxyethylene (20) sorbitan monolaurate),or the like; a chautropic agent, such as urea, formamide, guanidine, orthe like, and the anionic surfactant is preferable, and SDS isparticularly preferable.

Action of these protein denaturing agents on the complex pertaining tothe present invention, in (2-A), is generally carried out by bringinginto contacted the solution containing the protein denaturing agent(hereinafter, it may be abbreviated as “the protein denaturingagent-containing solution”, in some cases), and the complex of the Timprotein bound to the carrier pertaining to the present invention, andthe extracellular membrane vesicle or the virus in the sample to makeacted the protein denaturing agent-containing solution on the complexpertaining to the present invention.

It should be noted that the contacting of the protein denaturingagent-containing solution and the complex pertaining to the presentinvention can be carried out, for example, by a method for suspendingthe complex in the solution (when the carrier is the beads, and thelike); a method for dipping the complex into the solution (when thecarrier is a disc-like piece, a tube, or the like); a method for theaddition of the solution to the complex (the carrier), (when the carrieris the microplate, the tube, or the like).

—Solution for Containing Protein Denaturing Agent—

The solution for containing the protein denaturing agent, in (2-A),includes purified water, the buffer solution which is capable ofdissolving the protein denaturing agent, or the like. The buffersolution includes the buffer solution having buffer action at usually pH6 to 9, preferably 7 to 8 (for example, Tris, HEPES, or the like). Inaddition, buffer agent concentration in these solutions is appropriatelyselected from a range of usually 5 to 100 mM, and preferably 10 to 50mM.

—Protein Denaturing Agent-Containing Solution—

The protein denaturing agent-containing solution has a pH of usually 6.0to 9.0, and preferably 7.0 to 8.0, in (2-A). Concentration of theprotein denaturing agent in the protein denaturing agent-containingsolution is different depending on kind of the protein denaturing agent,and generally, may be within a concentration range to be used in thisfield, and for example, when using SOS, it is usually 0.1 to 10%,preferably 0.3 to 4%, and more preferably 0.5 to 2%. In addition, theprotein denaturing agent-containing solution may contain, for example,saccharides, salts, such as NaCl, and the like, a preservative, aprotein, or the like. Usually 10 μL to 500 μL, preferably 20 μL to 200μL, and more preferably 50 μL to 100 μL of the protein denaturingagent-containing solution, relative to 1 mg of the Tim carrier of thepresent invention is used, in (2-A).

—Action (Contact) Condition—

Temperature or time for bringing into acted (contacted) the proteindenaturing agent on the complex, in (2-A), is at usually 4.0 to 37° C.,preferably 10 to 30° C., and more preferably 20 to 30° C., for usually5.0 to 60 seconds, preferably 10 to 30 seconds, and more preferably 10to 20 seconds.

—Tim Protein—

As the Tim protein pertaining to the present invention in obtaining theextracellular membrane vesicle, in bringing into acted (contacted) theprotein denaturing agent (that is, in the (2-A)), any one may be used,as long as it is the Tim protein pertaining to the present invention,and, the Tim-4 protein pertaining to the present invention and the Tim-1protein pertaining to the present invention are preferable, and theTim-4 protein pertaining to the present invention is particularlypreferable, and in obtaining the virus, the Tim-4 protein pertaining tothe present invention and the Tim-3 protein pertaining to the presentinvention are particularly preferable.

Specific Example of (2-A)

(2-A) may be carried out, for example, by the following method. That is,the extracellular membrane vesicles or the viruses are separated fromthe complex pertaining to the present invention, after carrying out thecomplex formation step, the complex separation step, and carrying outthe washing operation, as needed, by the addition of usually 10 μL to500 μL, preferably 20 μL to 200 μL, and more preferably 50 μL to 100 μL,relative to 1 mg of the Tim carrier of the present invention, of thesolution containing usually 0.1 to 10%, preferably 0.3 to 4.0%, and morepreferably 0.5 to 2.0% of the protein denaturing agent (the solutioncontaining the protein denaturing agent in purified water, or the buffersolution which has buffer action at usually pH 6 to 9, and preferably 7to 8), to the resulting complex pertaining to the present invention, andby subjecting them to a reaction at usually 4 to 37° C., preferably 10to 30° C., and more preferably 20 to 30° C., for usually 5 to 60seconds, preferably 10 to 30 seconds, and more preferably 10 to 20seconds, while stirring by using a vortex mixer, or the like, so as tomake acted by bringing into contacted the Tim protein pertaining to thepresent invention in the complex pertaining to the present invention,and the protein denaturing agent.

<(2-B): Method for Decreasing Calcium Ion Concentration>

(2-B) is a method for separating the extracellular membrane vesicle orthe virus pertaining to the present invention from the complexpertaining to the present invention, by decreasing calcium ionconcentration bound to the resulting complex pertaining to the presentinvention, and the calcium ion in the solution containing the complex,after carrying out the complex formation step, the complex separationstep, and carrying out the washing operation, as needed.

In this way, the extracellular membrane vesicle or the virus pertainingto the present invention can be obtained in high purity and in an intactstate.

By the way, it has been known that the calcium ion is intermediated inbinding of the Tim protein and phosphatidyl serine (Immunity 2007December; 27 (6): 941-951). Co-presence of the calcium ion is requiredalso for the resulting complex pertaining to the present invention,after carrying out the complex formation step, and the complexseparation step, and the washing operation, as needed, to maintainbinding of the Tim protein pertaining to the present invention in thecomplex pertaining to the present invention, and the extracellularmembrane vesicle or the virus in the sample. It is necessary to maintain0.5 mM or more of the calcium ion in the solution of the complexpertaining to the present invention, for example, when the complexpertaining to the present invention is made co-present in the solution.Here, a pellet state carrier, after carrying out the complex formationstep, the complex separation step, and the washing operation, as needed,is also encompassed, when the complex pertaining to the presentinvention is made co-present in the solution.

It should be noted that the calcium ion never elutes from the complexunless the obtaining step is carried out, even if the complex pertainingto the present invention is dried, after carrying out the complexformation step, the complex separation step, and the washing operation,as needed, and thus binding of the carrier of the present invention andthe extracellular membrane vesicle or the virus is maintained.

It is possible, in (2-B), to separate the extracellular membrane vesicleor the virus pertaining to the present invention from the complexpertaining to the present invention, by decreasing concentration of thecalcium ion, entrained from the calcium ion bound to the complexpertaining to the present invention, to lower than requiredconcentration (effective concentration) to maintain binding of the Timprotein pertaining to the present invention and the extracellularmembrane vesicle or the virus, as described above.

Specifically, calcium ion concentration in the solution containing thecomplex pertaining to the present invention may be set usually lowerthan 0.5 mM, preferably lower than 0.4 mM, and more preferably lowerthan 0.2 mM.

The method for decreasing (effective) concentration of the calcium ionincludes, for example, the following (2-B-i), (2-B-ii), or the like.

(2-B-i) Method for using calcium ion chelating agent.

(2-B-ii) Method for using solution not containing calcium ion.

—(2-B-i) Method for Using Calcium Ion Chelating Agent.—

(2-B-i) is a method for separating the extracellular membrane vesicle orthe virus from the complex pertaining to the present invention, aftercarrying out the complex formation step, the complex separation step,and the washing operation, as needed, and after making acted the calciumion chelating agent on the calcium ion bound to the resulting complexpertaining to the present invention, and the calcium ion entrained fromthe solution containing the complex, by decreasing (effective)concentration of the calcium ion bound to the complex pertaining to thepresent invention, and the calcium ion entrained from the solutioncontaining the complex.

In this way, the extracellular membrane vesicle or the virus pertainingto the present invention can be obtained in high purity and in an intactstate.

—Calcium Ion Chelating Agent—

The calcium ion chelating agent may be any compound, as long as it iscapable of chelating the calcium ion, and includes, for example, EDTA(ethylenediamine tetraacetate), NTA (nitrilotriacetate), DTPA(diethylenetriamine pentaacetate), GLDA (L-glutamic acid diacetate),HEDTA (hydroxyethylethylenediamine triacetate), GE DTA (ethyleneglycolbis(β-aminoethyl ether)-N,N,N,N-tetraacetate), TTHA(triethylenetetramine-N,N, N′,N″,N′″,N′″-hexaacetate), HIDA(2-hydroxyethyliminodiacetic acid), DHEG(N,N-bis(2-hydroxtethyl)glycin), CyDTA(trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid monohydrate),or the like, and EDTA, GEDTA, and CyDTA are preferable. The solutioncontaining the calcium ion chelating agents (hereinafter it may beabbreviated as “the calcium ion chelating agent-containing solution”, insome cases) is generally brought into contacted with the pellet-likecomplex pertaining to the present invention, then the calcium ion boundto the complex pertaining to the present invention, and the calcium ionchelating agent in the calcium ion chelating agent-containing solutionare subjected to a reaction, to make acted these calcium ion chelatingagents to the calcium ion bound to the complex pertaining to the presentinvention.

It should be noted that contacting of the calcium ion chelatingagent-containing solution and the complex pertaining to the presentinvention can be carried out, for example, by a method for suspendingthe complex in the solution (when the carrier is the beads, and thelike); a method for dipping the complex in the solution (when thecarrier is disc-like piece, a tube, or the like); a method for theaddition of the solution to the complex (carrier) (when the carrier isthe microplate, the tube, or the like), or the like.

—Calcium Ion Chelating Agent-Containing Solution—

The solution for containing the calcium ion chelating agent, in (2-B-i),may be any one, as long as it dissolves the calcium ion chelating agent,and includes for example, purified water, a buffer solution, or thelike. As the buffer solution, the one which has buffer action at usuallypH 7.0 to 8.0, preferably 7.2 to 7.6 (for example, PBS, TBS, HBS or thelike) is preferable. In addition, buffer agent concentration in thesebuffer solutions is appropriately selected from a range of usually 5 to50 mM, and preferably 10 to 30 mM, and NaCl concentration isappropriately selected from a range of usually 100 to 200 mM, andpreferably 140 to 160 mM. For example, saccharides, salts, such as NaCl,and the like, a surfactant, a preservative, a protein, or the like, maybe contained in the calcium ion chelating agent-containing solution.

Concentration of the calcium ion chelating agent in the calcium ionchelating agent-containing solution, in (2-B-i), is usually 0.5 to 500mM, preferably 0.5 to 100 mM, and more preferably 0.5 to 50 mM.

The calcium ion chelating agent-containing solution, in (2-B-i), has apH of usually 6.0 to 9.0, preferably 7.0 to 8.0, and more preferably 7.2to 7.6.

Amount of the calcium ion chelating agent-containing solution to bemixed in the reaction solution, in (2-B-i), may be such amount thatconcentration of the calcium ion in the reaction solution attains lowerthan effective concentration, and the extracellular membrane vesicle orthe virus can be separated from the complex pertaining to the presentinvention.

—Acting (Contacting) Condition—

Temperature or time of bringing into acted (contacted) the calcium ionchelating agent on the complex, in (2-B-i), is usually 4.0 to 37° C.,preferably 10 to 30° C., more preferably 20 to 30° C., and usually 5 to60 seconds, preferably 10 to 30 seconds, and more preferably 10 to 20seconds.

—Tim Carrier—

Binding form of the Tim protein pertaining to the present invention andthe carrier pertaining to the present invention, in (2-B-i), is notlimited, as long as the carrier pertaining to the present invention andthe Tim protein pertaining to the present invention are bound. The onein which the carrier pertaining to the present invention binds to the SHgroup of the Tim protein pertaining to the present invention ispreferable, and the one in which the carrier pertaining to the presentinvention binds to the SH group of the Tim-4 protein pertaining to thepresent invention is particularly preferable, when the extracellularmembrane vesicle or the virus is eluted by using the calcium ionchelating agent (that is, in (2-B-i)), because many extracellularmembrane vesicle or virus can be obtained.

Specific Example of (2-B-i)

(2-B-i) may be carried out, for example, by the following method. Thatis, the extracellular membrane vesicle or the virus is separated fromthe complex pertaining to the present invention, after carrying out thecomplex formation step, the complex separation step, and carrying outthe washing operation, as needed, by the addition of usually 10 μL to500 μL, preferably 20 μL to 200 μL, and more preferably 50 μL to 100 μLof the solution containing usually 0.5 to 500 mM, preferably 0.5 to 100mM, and more preferably 0.5 to 50 mM of the calcium ion chelating agent(a solution containing the calcium ion chelating agent, in purifiedwater, or the buffer solution which has buffer action at usually pH 7.0to 8.0, preferably 7.2 to 7.6), to the resulting complex pertaining tothe present invention, relative to 1 mg of the Tim carrier of thepresent invention, and by subjecting them to a reaction at usually 4.0to 37° C., preferably 10 to 30° C., and more preferably 20 to 30° C.,for usually 5 to 60 seconds, preferably 10 to 30 seconds, and morepreferably 10 to 20 seconds, while stirring by using a vortex mixer, orthe like.

—(2-B-ii) Method for Using Solution not Containing Calcium Ion—

(2-B-ii) is a method for separating the extracellular membrane vesicleor the virus from the complex pertaining to the present invention, aftercarrying out the complex formation step, the complex separation step,and the washing operation, as needed, by bringing into contacted theresulting complex pertaining to the present invention to the solutionnot containing the calcium ion to decrease (dilute) (effective)concentration of the calcium ion bound to the complex pertaining to thepresent invention.

That is, the extracellular membrane vesicle can be separated from thecomplex pertaining to the present invention, by decreasing (diluting)(effective) concentration of the calcium ion, which is required tomaintain binding of the Tim protein pertaining to the present inventionin the complex pertaining to the present invention, and theextracellular membrane vesicle or the virus in the sample, by bringinginto contacted the solution not containing the calcium ion to thecomplex pertaining to the present invention.

It should be noted that contact of the solution not containing thecalcium ion and the complex pertaining to the present invention can becarried out, for example, by a method for suspending the complex in thesolution (when the carrier is the beads, or the like); a method fordipping the complex in the solution (when the carrier is a disc-likepiece, a tube, or the like); a method for the addition of the solutionto the complex (carrier) (when the carrier is the microplate, the tube,or the like), or the like.

—Solution not Containing Calcium Ion—

The solution not containing the calcium ion to be added to the resultingcomplex pertaining to the present invention, in (2-B-ii), may be anyone, as long as not denaturing the extracellular membrane vesicle or thevirus, and includes for example, purified water, the buffer solution, orthe like. As the buffer solution, the one which has buffer action atusually pH 7.0 to 8.0, preferably 7.2 to 7.6 (for example, PBS, TBS, HBSor the like) is preferable. In addition, buffer agent concentration inthese buffer solutions is appropriately selected from a range of usually5 to 50 mM, and preferably 10 to 30 mM, and NaCl concentration isappropriately selected from a range of usually 100 to 200 mM, andpreferably 140 to 160 mM. For example, saccharides, salts, such as NaCl,and the like, a surfactant, a preservative, a protein, or the like, maybe contained in the solvent not containing the calcium ion.

Amount of the solution not containing the calcium ion to be added to theresulting complex pertaining to the present invention, in (2-B-ii), maybe any amount, as long as it is capable of making calcium ionconcentration lower than the effective concentration.

Specific Example of (2-B-ii)

(2-B-ii) is carried out by the addition of the solution not containingthe calcium ion to the resulting complex pertaining to the presentinvention, after carrying out the complex formation step, the complexseparation step, and as needed, after carrying out the washingoperation, in such an amount that attains a calcium ion concentration ofusually lower than 0.5 mM, preferably lower than 0.1 mM, more preferablylower than 0.01 mM, in the solution containing the complex pertaining tothe present invention.

It should be noted that, the solution containing the calcium ion may bereplaced to the solution not containing the calcium ion and/or dilutedby the solution not containing the calcium ion, after carrying out thecomplex formation step, the complex separation step, and after carryingout the washing operation, as needed, so as to attain the finalconcentration of the calcium ion to lower than the effectiveconcentration, when the resulting complex pertaining to the presentinvention is present in the solution containing the calcium ion (forexample, a reaction solution after carrying out the complex formationstep, or a calcium ion-containing washing solution).

(2-B-i) is preferable, because amount (volume increase) of the solutioncontaining the resulting extracellular membrane vesicle by the obtainingstep is more in (2-B-ii) than in (2-B-i).

In the above description, the solution brought into contacted andinteracted with the complex (the protein denaturing agent-containingsolution, the calcium ion chelating agent-containing solution, thesolution not containing the calcium ion) results in to contain thecarrier and extracellular membrane vesicle or the virus separated(released) from the carrier (complex). Therefore, the solutioncontaining the extracellular membrane vesicle or the virus can beobtained by removing the carrier from the solution, and recovering onlythe solution.

The extracellular membrane vesicle or the virus can be efficientlyremoved from the sample, therefore, the sample with less contaminantscan be obtained by using the Tim carrier of the present invention.

The removing method of the present invention is capable of removing theextracellular membrane vesicle or the virus in the sample in goodprecision and efficiently, which cannot be removed completely byconventional methods, such as ultracentrifugal separation method,polymer sedimentation treatment, and the like.

<7. Removing Method of Extracellular Membrane Vesicle or the Virus inSample>

The method for obtaining the extracellular membrane vesicle or the virusof the present invention (hereinafter it may be abbreviated as “theremoving method of the present invention”, in some cases) ischaracterized by including the following steps.

(1) The step for forming the complex of the Tim protein bound to thecarrier, and the extracellular membrane vesicle or the virus in thesample, in the presence of the calcium ion (hereinafter, it may beabbreviated as “the complex formation step”, in some cases).(2) The step for separating the complex and the sample (hereinafter, itmay be abbreviated as “the complex separation step”, in some cases).<7-1. Complex Formation Step>

The complex formation step in the removing method of the presentinvention is the same as the complex formation step in the obtainingstep of the present invention, and various kinds of preferableconditions are also the same.

<7-2. Complex Separation Step>

The complex separation step in the removing method of the presentinvention is a step for separating the resulting complex of the Timprotein bound to the carrier pertaining to the present invention (theTim carrier of the present invention), and the extracellular membranevesicle or the virus in the sample (the complex pertaining to thepresent invention), and the sample, after carrying out the complexformation step, to obtain the separated sample.

By carrying out this step, the sample from where the extracellularmembrane vesicle or the virus is removed can be obtained.

The complex separation step in the removing method of the presentinvention may be any method, as long as it is capable of removing thecomplex pertaining to the present invention from the sample, andincludes the same method as the complex separation step in the obtainingmethod of the present invention. After separating the complex of thepresent invention and the sample in this way, the separated sample maybe obtained (recovered) by a method known per se.

In this way, the extracellular membrane vesicle or the virus in thesample can be removed in good precision and efficiently, which cannot beremoved completely by a conventional method, such as theultracentrifugal separation method or a polymer precipitation treatment,or the like.

In addition, removal of the extracellular membrane vesicle or the virusfrom the sample can be carried out more efficiently, by repeating thecomplex formation step and the complex separation step multiple times.Either a new carrier may be used, or a carrier after use may also bere-utilized, in carrying out the complex formation step and the complexseparation step repeatedly. In re-utilization of the carrier, thecomplex pertaining to the present invention, which was removed at thecomplex separation step in the removing method of the present invention,may be subjected to the same treatment as the obtaining step of theobtaining method of the present invention ((2-B) a method for decreasingcalcium ion concentration) using the solution containing the calcium ionchelating agent, or the solution not containing the calcium ion. In thisway, the Tim carrier of the present invention and the extracellularmembrane vesicle or the virus can be separated from the complexpertaining to the present invention. That is, removal of theextracellular membrane vesicle or the virus in the sample can be carriedout in good precision and efficiently, by re-utilization of the Timcarrier of the present invention, because the Tim carrier of the presentinvention can be used again in the removing method of the presentinvention.

The extracellular membrane vesicle or the virus in the sample can bedetected in good precision and in high sensitivity, by using the Timcarrier of the present invention.

<8. Method for Detecting Extracellular Membrane Vesicle or the Virus inSample>

A method for detecting the extracellular membrane vesicle or the virusof the present invention (hereafter it may be abbreviated as “thedetection method of the present invention”, in some cases) ischaracterized by comprising the following steps:

(1) A step for forming the complex of the Tim protein bound to thecarrier, and the extracellular membrane vesicle or the virus in thesample, in the presence of the calcium ion (complex formation step).

(2) A step for detecting the complex (detection step).

<8-1. Complex Formation Step>

The complex formation step in the detection method of the presentinvention is a step for forming the complex of the Tim carrier of thepresent invention, and the extracellular membrane vesicle or the virusin the sample, in the presence of the calcium ion.

—Sample Pertaining to the Present Invention—

The sample pertaining to the present invention is the same as the samplein the obtaining method of the present invention.

—Calcium Ion Concentration and Origin of Calcium Ion—

In the detection method of the present invention, the calcium ion ispresent in forming the complex of the Tim carrier pertaining to thepresent invention, and the extracellular membrane vesicle or the virusin the sample (the complex pertaining to the present invention).

In the detection method of the present invention, calcium ionconcentration, in bringing into contacted the Tim carrier pertaining tothe present invention, and the extracellular membrane vesicle or thevirus in the sample pertaining to the present invention, is usually 0.5to 100 mM, preferably 1 to 10 mM, and more preferably 2 to 5 mM. Itshould be noted that it is not without saying that the calcium ion ofsuch concentration as described above is necessary in the solutioncontaining the complex, until the complex of the Tim carrier pertainingto the present invention, and the extracellular membrane vesicle or thevirus in the sample pertaining to the present invention is formed andthe detection step is carried out, that is, until the complex issubjected to the detection step.

In addition, origin of the calcium ion is not especially limited, andincludes the same as specific example as origin of the calcium ion inthe obtaining method of the present invention.

It should be noted that as a method for the calcium ion to be present inbringing into contacted the Tim carrier of the present invention, andthe extracellular membrane vesicle or the virus in the sample, generallythe calcium ion as described above may be contained in the sample, insuch an amount that attains a calcium ion concentration of the aboverange, in bringing into contacted the Tim carrier of the presentinvention, and the extracellular membrane vesicle or the virus in thesample.

The solution for containing the calcium ion is the same as the solutionfor containing (dissolving or suspending) the extracellular membranevesicle or the virus pertaining to the present invention, and specificexamples, and the like, are also the same.

—Amount of Sample—

Amount of the sample, in the complex formation step of the detectionmethod of the present invention, is usually 0.1 to 1000 mL, preferably0.1 to 500 mL, and more preferably 0.1 to 100 mL, relative to 1 mg ofthe Tim carrier of the present invention, in using the beads as thecarrier pertaining to the present invention, and is usually 50 μL to 300μL, and preferably 100 μL to 200 μL, relative to 1 well, in using themicroplate as the carrier pertaining to the present invention.

—Temperature—

In the complex formation step of the detection method of the presentinvention, temperature in bringing into contacted the Tim carrier of thepresent invention, and the extracellular membrane vesicle or the virusin the sample, is usually 2 to 37° C., and preferably 2 to 30° C.

—Time—

In the complex formation step of the detection method of the presentinvention, contacting time of the Tim protein pertaining to the presentinvention and the sample is usually 0.5 to 24 hours, preferably 1 to 20hours, and more preferably 1 to 12 hours.

—Tim Protein—

As the Tim protein pertaining to the present invention in the detectionmethod of the present invention, any one may be used, as long as beingthe Tim protein pertaining to the present invention, however, the Tim-4protein pertaining to the present invention is particularly preferable.

—Tim Carrier—

Binding form of the Tim protein pertaining to the present invention andthe carrier pertaining to the present invention is not especiallylimited, as long as the carrier pertaining to the present invention andthe Tim protein pertaining to the present invention are bound, and, theone where the carrier pertaining to the present invention is bound tothe SH group of the Tim protein pertaining to the present invention ispreferable, and the one where the carrier pertaining to the presentinvention is bound to the SH group of the Tim-4 protein pertaining tothe present invention is particularly preferable, because they arecapable of detecting the extracellular membrane vesicles or the virusesin high sensitivity.

Specific Example of Complex Formation Step in Detection Method of thePresent Invention

The complex formation step in the detection method of the presentinvention may be carried out, for example, by the following method.

That is, in using the beads as the carrier pertaining to the presentinvention, the complex of the Tim protein bound to the carrierpertaining to the present invention, and the extracellular membranevesicles or the viruses in the sample is formed, by bringing intocontacted usually 0.001 to 20 mg, preferably 0.005 to 10 mg, and morepreferably 0.01 to 6.0 mg of the Tim carrier of the present invention,relative to 1 mL of the solution after mixing the Tim carrier of thepresent invention, and the solution containing the sample and thecalcium ion (the solution in forming the complex pertaining to thepresent invention); the solution containing the calcium ion, in such anamount that attains a concentration of the calcium ion of usually 0.5 to100 mM, preferably 1.0 to 10 mM, and more preferably 2.0 to 5.0 mM, inthe solution in forming the complex pertaining to the present invention;and usually 0.1 to 1000 mL, preferably 0.1 to 500 mL, and morepreferably 0.1 to 100 mL of the sample, relative to 1 mg of the Timcarrier of the present invention; at usually 2 to 37° C., and preferably2 to 30° C., for usually 0.5 to 24 hours, preferably 1 to 20 hours, andmore preferably 1 to 12 hours.

In using the microplate as the carrier pertaining to the presentinvention, the complex of the Tim carrier of the present invention, andthe extracellular membrane vesicles or the viruses in the sample (thecomplex pertaining to the present invention) is formed by the additionof the solution containing the calcium ion, in such an amount thatattains a calcium ion concentration of usually 0.5 to 100 mM, preferably1.0 to 10 mM, and more preferably 2.0 to 5.0 mM, in the solution informing the complex pertaining to the present invention, and usually 50μL to 300 μL, and preferably 100 μL to 200 μL of the sample pertainingto the present invention, relative to 1 well, to each well of themicroplate on which the Tim protein is immobilized (the Tim carrier ofthe present invention) to be subjected to a reaction at usually 2° C. to37° C., and preferably 2° C. to 30° C., for usually 0.5 hour to 24hours, preferably 1 hour to 20 hours, and more preferably 1 to 12 hours.

<8-2. Detection Step in Detection Method of the Present Invention>

The detection step in the detection method of the present invention is astep for detecting the complex of the Tim carrier of the presentinvention, and the extracellular membrane vesicle or the virus in thesample (the complex pertaining to the present invention), by carryingout the complex separation step of the sample, as needed, after carryingout the complex formation step, and after carrying out the washingoperation, as needed.

—Complex Separation Step—

The complex separation step in the detection method of the presentinvention is, after the complex formation step, a step for removing thesample from the resulting complex of the Tim protein bound to thecarrier pertaining to the present invention (the Tim carrier of thepresent invention), and the extracellular membrane vesicle or the virusin the sample (the complex pertaining to the present invention), asneeded.

The complex separation step in the detection method of the presentinvention may be any method, as long as it is capable of separating thecomplex pertaining to the present invention and the sample, in otherwords, is capable of what is called B/F separation, and the B/Fseparation method usually used in this field can be used. Such a methodincludes the same one as the complex separation step in the obtainingmethod of the present invention.

Specific Example of Complex Separation Step

The complex separation step in the detection method of the presentinvention may be carried out, according to a usual method in this field,and, in using the magnetic beads as the carrier pertaining to thepresent invention, for example, a container used in the complexformation step is placed on a magnet stand, as needed, to collect thecomplex pertaining to the present invention on the tube wall usingmagnetic force, to remove the sample of the supernatant.

In using the microplate as the carrier pertaining to the presentinvention, the sample is removed using a micropipette or a plate washer,as need, for example, from the microplate after carrying out the complexformation step.

<8.3. Washing Operation>

After the complex formation step, and carrying out the complexseparation step, as needed, the resulting complex pertaining to thepresent invention may be washed further using a washing solutioncontaining the calcium ion (hereafter it may be abbreviated as “washingoperation”, in some cases), as needed. Impurities in the samplepertaining to the present invention, such as cell-derived components,and the like, adhered at the carrier surface pertaining to the presentinvention can be removed by the washing operation. Various conditions ofthe washing method, and the like, are the same as those in the washingoperation in the obtaining method of the present invention.

Specific Example of Washing Operation

In carrying out the washing operation in a tube having a volume of 1.5mL, using the magnetic beads as the carrier pertaining to the presentinvention, firstly, usually 100 μL to 1500 μL, and preferably 200 μL to1000 μL of the washing solution containing the calcium ion (a buffersolution, containing, for example, usually 0.5 to 100 mM, preferably 1to 10 mM, and more preferably 2 to 5 mM of the calcium ion, and havingbuffering action at pH 7.0 to 8.0, and preferably 7.2 to 7.6, and notprecipitating calcium. However, the phosphate buffer is not preferablebecause it precipitates by binding with calcium.) is added into acontainer containing the resulting complex pertaining to the presentinvention by the complex separation step, and stirred. After that, thecontainer is placed at the magnet stand to collect the complex on thetube wall, using magnetic force, and the solution in the container isdiscarded. The washing operation may be carried out repeatedly severaltimes, as needed.

In using the microplate as the carrier pertaining to the presentinvention, firstly, usually 100 μL to 300 μL, and preferably 200 μL to300 μL of the calcium ion-containing washing solution (for example, thebuffer solution containing usually 0.5 to 100 mM, preferably 1 to 10 mM,and more preferably 2.0 to 5.0 mM of the calcium ion, and havingbuffering action at pH 7.0 to 8.0, and preferably 7.2 to 7.6, and notprecipitating calcium. However, the phosphate buffer is not preferablebecause it precipitates by binding with calcium.) is added in a wellcontaining the resulting complex pertaining to the present invention bythe complex separation step, and then the added washing solution isremoved. The washing operation may be carried out repeatedly severaltimes, as needed.

<8-4. Detection Step>

The detection step in the detection method of the present invention maybe any method, as long as it is a method capable of detecting presenceor absence and/or amount of the complex pertaining to the presentinvention, and any method known per se to be used for immunologicalmeasurement can be used.

The detection step of the present invention is not especially limited,except for using the Tim carrier of the present invention prepared bythe method as described above. Such an immunological measurement methodincludes all of, for example, a measurement method utilizing anagglutination reaction, such as a reversed passive agglutinationreaction method (“(sequel) Biochemical Experimental Course 5,Immuno-biochemical Research Method” p. 36-37, Tokyo Kagaku Dojin Co.,Ltd.; “Kanai's Manual of Clinical Laboratory Medicine”, 30^(th) edition,p. 844-845, Kanehara Publishing Co., Ltd., or the like.), for example,an optical measurement method applying the agglutination reaction, suchas nephelometry (“Kanai's Manual of Clinical Laboratory Medicine”,30^(th) edition, p. 851-853, Kanehara Publishing Co., Ltd., or thelike.), immunoturbidimetry (“Kanai's Manual of Clinical LaboratoryMedicine”, 30^(th) edition, p. 853-854, Kanehara Publishing Co., Ltd.,or the like.), radioimmunoassay (RIA) (“(sequel) BiochemicalExperimental Course 5, Immuno-biochemical Research Method” p. 57-61,Tokyo Kagaku Dojin Co., Ltd.; “Kanai's Manual of Clinical LaboratoryMedicine”, 30^(th) edition, p. 856-862, Kanehara Publishing Co., Ltd.,or the like.), immune-radiometric assay (IRMA) (“Kanai's Manual ofClinical Laboratory Medicine”, 30^(th) edition, p. 856-862, KaneharaPublishing Co., Ltd., or the like.), enzyme immunoassay (EIA) (“(sequel)Biochemical Experimental Course 5 Immuno-biochemical Research Method” p.62-65, Tokyo Kagaku Dojin Co., Ltd.; “Kanai's Manual of ClinicalLaboratory Medicine”, 30^(th) edition, p. 862-865, Kanehara PublishingCo., Ltd.; JP-A-56-106154; JP-A-58-23796, or the like.), solid phaseenzyme immunoassay (ELISA) (“Kanai's Manual of Clinical LaboratoryMedicine”, 30^(th) edition, p. 1145-1149, Kanehara Publishing Co., Ltd.,or the like.), fluorescence/luminescence immunoassay (“Kanai's Manual ofClinical Laboratory Medicine”, 30^(th) edition, p. 865-867, KaneharaPublishing Co., Ltd., or the like.), the flow cytometry method, or thelike, and the solid phase enzyme immunoassay (ELISA) or the flowcytometry is preferable.

When the detection step of the present invention is carried out by thesolid phase enzyme immunoassay (ELISA) or the flow cytometry, it may becarried out, according to a method known per se, as long as the complexpertaining to the present invention is a detection object. There isincluded, for example, a method for using a labeled primary antibodywhich is a primary antibody labeled with a labelling substance, such asan anti-extracellular membrane vesicle antibody or an anti-virusantibody capable of binding with the extracellular membrane vesicle orthe virus, or a method for using the primary antibody and a labeledsecondary antibody that binds to the primary antibody. Any labeledprimary antibody and labeled secondary antibody may be used, as long asthey are used for a labeled antibody of the ELISA method or the flowcytometry method, and includes, for example, a fluorescent labeledantibody labeled with a fluorescent substance, such as Cy3, Cy5, FITC,rhodamine, PE or the like, an enzyme labeled antibody labeled with anenzyme, such as a peroxidase, an alkaline phosphatase, or the like, themagnetic beads labeled antibody, an infrared labeled antibody, or thelike. Fluorescence, or the like, pertaining to these labeled antibodiesmay be measured by a method known per se, corresponding to the labelingmethod (labeling substance) of the labeled antibody.

—Diluent of Labelled Antibody—

A diluent of the labelled primary antibody, or the primary antibody andthe labelled secondary antibody to be subjected to a reaction with thecomplex pertaining to the present invention, in the detection method ofthe present invention, may be any one, as long as it does not inhibitbinding of the complex of the Tim protein and the extracellular membranevesicle or the virus in the sample, and the antibody, except forcontaining the calcium ion, and includes, for example, water, or thebuffer solution which has buffer action at pH 7.0 to 8.0, and preferably7.2 to 7.6 (for example, TBS, HBS or the like), or the like. It shouldbe noted that the phosphate buffer is not preferable, because itprecipitates by binding with calcium. In addition, buffer concentrationin theses buffer solutions is appropriately selected from a range ofusually 5 to 50 mM, and preferably 10 to 30 mM, and NaCl concentrationis appropriately selected from a range of usually 100 to 200 mM, andpreferably 140 to 160 mM. For example, saccharides, salts such as NaCl,and the like, a surfactant, a preservative, a protein, or the like, maybe contained in these solutions, as long as in such amount that does notinhibit binding of the complex of the Tim carrier pertaining to thepresent invention and the extracellular membrane vesicle or the virus inthe sample. The surfactant includes, for example, Tween 20, andsurfactant concentration in the solution for containing theextracellular membrane vesicle or the virus relevant to the presentinvention is usually 0.00001% to 0.2%, and preferably 0.0005% to 0.1%.In addition, concentration of the calcium ion contained in the diluentis usually 0.5 to 100 mM, preferably 1 to 10 mM, and more preferably 2to 5 mM.

In the detection method of the present invention, dilution degree of thelabeled primary antibody or the primary antibody and the labeledsecondary antibody to be subjected to a reaction with the complexpertaining to the present invention, is different depending on activityor concentration of the antibody, however, it is usually 10-fold to1000000-fold, and preferably 1000-fold to 100000-fold.

In the detection method of the present invention, amount of the diluentof the labeled primary antibody, or the primary antibody and the labeledsecondary antibody to be subjected to a reaction with the complexpertaining to the present invention, is usually 0.1 mL to 1000 mL,preferably 0.1 mL to 500 mL, and more preferably 0.1 mL to 100 mL,relative to 1 mg of the carrier pertaining to the present invention,when the carrier pertaining to the present invention is the beads; andit is usually 50 μL to 300 μL, preferably 50 μL to 200 μL, and morepreferably 50 μL to 100 μL, relative to 1 well, when the carrierpertaining to the present invention is the microplate.

—Reaction (Contacting) Temperature—

In the detection method of the present invention, reaction temperatureof the complex pertaining to the present invention, and the labelledprimary antibody, or the primary antibody and the labelled secondaryantibody, is usually 2 to 37° C., preferably 11 to 337° C., and morepreferably 20 to 30° C.

—Reaction (Contacting) Time—

In the detection method of the present invention, reaction time of thecomplex pertaining to the present invention and the labelled primaryantibody, or the primary antibody and the labelled secondary antibody,is usually 0.5 to 12 hours, preferably 1 to 4 hours, and more preferably1 to 2 hours.

It is possible to remove the unreacted labelled primary antibody, by thewashing operation after subjecting the complex pertaining to the presentinvention and labelled primary antibody to a reaction, when the labelledprimary antibody is used in the detection operation of the detectionstep in the detection method of the present invention. In addition, itis possible to remove the unreacted primary antibody and the labelledsecondary antibody, by the washing operation after subjecting thecomplex pertaining to the present invention and the primary antibody toa reaction, or after subjecting the complex pertaining to the presentinvention and the complex of primary antibody and the labelled secondaryantibody to a reaction, when the primary antibody and the labelledsecondary antibody are used. As the washing method, a washing methodusually used in this field can be used, except for using the washingsolution containing the calcium ion, as described above. The washingsolution containing the calcium ion, to be used in the washingoperation, may be any solution, as long as it contains usually 0.5 to100 mM, preferably 1 to 10 mM, and more preferably 2 to 5 mM of thecalcium ion, and does not inhibit binding of the extracellular membranevesicles or the viruses pertaining to the present invention in thecomplex, the Tim protein pertaining to the present invention, and thecarrier of the present invention, and includes for example, a buffersolution containing usually 0.5 to 100 mM, preferably 1 to 10 mM, andmore preferably 2 to 5 mM of the calcium ion, and having buffer actionat pH 7.0 to 8.0, and preferably 7.2 to 7.6, and not precipitatingcalcium (for example, TBS, HBS). It should be noted that the phosphatebuffer is not preferable, because it precipitates by binding withcalcium. In addition, buffer agent concentration in these buffersolutions is appropriately selected from a range of usually 5 to 50 mM,and preferably 10 to 30 mM, and NaCl concentration is appropriatelyselected from a range of usually 100 to 200 mM, and preferably 140 to160 mM. This solution may contain, for example, saccharides, salts suchas NaCl, and the like, a surfactant, a preservative, a protein, or thelike, as long as in such an amount that does not inhibit binding of theextracellular membrane vesicles or the viruses pertaining to the presentinvention in the complex, the Tim protein pertaining to the presentinvention and the carrier of the present invention. The surfactantincludes, for example, Tween 20 (produced by Wako Pure ChemicalIndustries Co.), or the like, and surfactant concentration in thewashing solution is usually 0.00001 to 0.2%, and preferably 0.0005 to0.1%. When the carrier pertaining to the present invention is the beads,it is usually 0.1 mL to 1000 mL, and preferably 0.1 mL to 500 mL, andmore preferably 0.1 mL to 100 mL, relative to 1 mg of the carrierpertaining to the present invention. Amount of the washing solution tobe used in 1 well is usually 100 μL to 300 μL, and preferably 200 μL to300 μL, and the washing solution is removed after the addition, when thecarrier pertaining to the present invention is the microplate. Thewashing operation may be repeated plural times, as needed.

—Detection—

The labelled substance of the labelled primary antibody or the labelledsecondary antibody includes peroxidase, alkali phosphatase, or the like,when the detection step in the detection method of the present inventionis chromogenic detection. These labelled substances may be detected,according to the known method per se, in response to each labelledsubstance.

The chromogenic substrate solution in the chromogenic detection may beany one, as long as it is a chromogenic substrate solution usually usedin this field, and includes, when peroxidase is used as the labelledsubstance, for example, a TMB (tetramethyl benzidine) solution or an OPD(o-phenylene diaime) solution, and preferably includes the TMB solution.

Amount of the chromogenic substrate in the chromogenic detection isusually 0.1 mL to 1000 mL, preferably 0.1 mL to 500 mL, and morepreferably 0.1 mL to 100 mL, relative to 1 mg of the carrier pertainingto the present invention, when the carrier pertaining to the presentinvention is the beads, and it is usually 50 μL to 300 μL, preferably 50μL to 200 μL, and more preferably 50 μL to 100 μL, relative to 1 well,when the carrier pertaining to the present invention is the microplate.

Reaction time with the chromogenic substance is usually 5 minutes to 60minutes, and preferably 10 minutes to 40 minutes, when the detectionstep in the detection method of the present invention is the chromogenicdetection.

Temperature of a reaction with the chromogenic substrate solution isusually 2° C. to 37° C., and preferably 20° C. to 30° C., when thedetection step in the detection method of the present invention is thechromogenic detection.

As a reaction stopping solution to stop the chromogenic reaction, astrong acid, such as usually 1 mol/L of hydrochloric acid, or 1 mol/L ofsulfuric acid, or the like, is added in the same amount as thechromogenic substrate solution to stop the chromogenic reaction, whenthe detection step in the detection method of the present invention isthe chromogenic detection.

Fluorescence is measured by the addition of a fluorescence measurementsolution to the complex of the complex pertaining to the presentinvention and the labelled primary antibody, or the complex of thecomplex pertaining to the present invention and the primary antibody andthe labelled secondary antibody, when the detection step in thedetection method of the present invention is fluorescence detection, anda fluorescence substance is used as the labelled substance of labelledprimary antibody or the labelled secondary antibody. Any of thefluorescence measurement solution usually used in this field(hereinafter, it may be abbreviated as “measuring solution”, in somecases) may be used, except for using the calcium ioncontaining-fluorescence measurement solution, as the fluorescencemeasurement solution. The calcium ion-containing measurement solution tobe used in the fluorescence measurement solution may be any one, as longas it contains usually 0.5 to 100 mM, preferably 1 to 10 mM, and morepreferably usually 2 to 5 mM of the calcium ion, and does not affectbinding of the extracellular membrane vesicle or the virus pertaining tothe present invention in the complex, the Tim protein pertaining to thepresent invention, and the carrier of the present invention, andincludes, for example, the buffer solution containing usually 0.5 to 100mM, preferably 1 to 10 mM, and more preferably 2 to 5 mM of the calciumion, and having buffer action at pH 7.0 to 8.0, and preferably 7.2 to7.6, and not precipitating calcium (for example, TBS, HBS). It should benoted that the phosphate buffer is not preferable because itprecipitates by binding with calcium. In addition, buffer agentconcentration in these buffer solutions is appropriately selected from arange of usually 5 to 50 mM, preferably 10 to 30 mM, and NaClconcentration is appropriately selected from a range of usually 100 to200 mM, preferably 140 to 160 mM. This solution may contain, forexample, saccharides, salts, such as NaCl, and the like, a surfactant, apreservative, a protein, such as BSA, as long as in such an amount thatdoes not inhibit binding of the extracellular membrane vesicle or thevirus pertaining to the present invention in the complex, the Timprotein pertaining to the present invention, and the carrier of thepresent invention. The surfactant includes, for example, Tween 20(produced by Wako Pure Chemical Industries Co.), or the like, andsurfactant concentration in the washing solution is usually 0.00001 to0.2%, and preferably 0.0005 to 0.1%. Amount of the fluorescencemeasurement solution to be used, in 1 well, in the detection method ofthe present invention is usually 50 μL to 300 μL, and preferably 50 μLto 200 μL, when the detection method of the present invention is theELISA method, and the carrier pertaining to the present invention is themicroplate. It is usually 0.1 mL to 1000 mL, preferably 0.1 mL to 500mL, more preferably 0.1 mL to 100 mL, relative to 1 mg of the carrierpertaining to the present invention, when the detection methodpertaining to the present invention is the flow cytometry method, andthe carrier pertaining to the present invention is the beads.

Specific Example of Detection Step in Detection Method of the PresentInvention

When the detection step is carried out by the ELISA method, using, forexample, the microplate, as the carrier pertaining to the presentinvention, after the complex formation step, and the complex separationstep, as needed, and further after carrying out the washing operation,as needed, to each well of the microplate, on which the resulting Timprotein pertaining to the present invention is immobilized, usually 50μL to 300 μL, preferably 50 μL to 200 μL, and more preferably 50 μL to100 μL of a diluted solution, which was obtained by diluting aperoxidase labeled primary antibody or a unlabeled primary antibody,against the extracellular membrane vesicles or the viruses, usingusually 10 times to 1000000 times, and preferably 1000 times to 100000times of a diluent containing usually 0.5 to 100 mM, preferably 1 to 10mM, and more preferably 2 to 5 mM of the calcium ion, is added relativeto 1 well, and subjected to a reaction at usually 2 to 37° C., andpreferably 11 to 37° C., for usually 0.5 to 12 hours, and preferably 1to 4 hours, and next each well is washed using a washing buffercontaining the calcium ion (a solution which contains usually 0.5 to 100mM, preferably 1 to 10 mM, and more preferably 2 to 5 mM of the calciumion, and does not influence binding of the extracellular membranevesicles or the viruses pertaining the present invention in the complex,the Tim protein pertaining to the present invention, and the carrier ofthe present invention). In using the unlabeled primary antibody, eachwell is washed using usually 100 μL to 300 μL, and preferably 200 μL to300 μL of the washing buffer containing the calcium ion (the solutionwhich contains usually 0.5 to 100 mM, preferably 1 to 10 mM, and morepreferably 2 to 5 mM of the calcium ion, and does not influence bindingof the extracellular membrane vesicles or the viruses pertaining to thepresent invention in the complex, the Tim protein pertaining to thepresent invention, and the carrier of the present invention), afterfurther subjecting the peroxidase labeled second antibody to a reactionwith the primary antibody by the same method. Next, usually 50 μL to 300μL, preferably 50 μL to 200 μL, and preferably 50 μL to 100 μL of achromogenic substrate solution, such as a TMB solution, an OPD solution,or the like, relative to 1 well, is added to each well and subjectedthem to a reaction at usually 2° C. to 37° C., and preferably 20° C. to30° C., for usually 5 minutes to 60 hours, and preferably 10 minutes to40 hours. After that, a reaction termination solution such as 1 mol/Lhydrochloric acid or 1 mol/L sulfuric acid, or the like, is added in thesame amount as the chromogenic substrate solution to terminate a colorreaction, and measure absorbance using a microplate reader.

When the detection step is carried out by the flow cytometry methodusing, for example, the beads, as the carrier pertaining to the presentinvention, after the complex formation step, and the complex separationstep, as needed, and further after carrying out the washing operation,as needed, to the beads, on which the resulting Tim protein pertainingto the present invention is immobilized, usually 0.1 mL to 1000 mL,preferably 0.1 mL to 500 mL, and more preferably 0.1 mL to 100 mL of thediluted solution, which was obtained by diluting a fluorescent labelledprimary antibody, or a unlabeled primary antibody, using usually 10times to 1000000 times, and preferably 1000 times to 100000 times of thediluent containing usually 0.5 to 100 mM, preferably 1 to 10 mM, andmore preferably 2 to 5 mM of the calcium ion, is added, relative to 1 mgof the carrier pertaining to the present invention, and subjected themto a reaction at usually 2 to 37° C., and preferably 11 to 37° C., forusually 0.5 to 12 hours, and preferably 1 to 4 hours, and next the beadsare washed using the washing buffer containing the calcium ion (thesolution which contains usually 0.5 to 100 mM, preferably 1 to 10 mM,and more preferably 2 to 5 mM of the calcium ion, and does not influencebinding of the extracellular membrane vesicles or the viruses pertainingto the present invention in the complex, the Tim protein pertaining tothe present invention, and the carrier of the present invention). Inusing the unlabeled primary antibody, the beads are washed using thewashing buffer containing the calcium ion (the solution which containsusually 0.5 to 100 mM, preferably 1 to 10 mM, and more preferably 2 to 5mM of the calcium ion, and does not influence binding of theextracellular membrane vesicles or the viruses pertaining to the presentinvention in the complex, the Tim protein pertaining to the presentinvention, and the carrier of the present invention), after furthersubjecting a fluorescent labelled second antibody to a reaction with theprimary antibody by the same method. Next, after suspending the beads bythe addition of usually 0.1 mL to 1000 mL, preferably 0.1 mL to 500 mL,and preferably 0.1 mL to 100 mL of a fluorescence measurement solution,relative to 1 mg of the carrier pertaining to the present invention,fluorescence intensity is measured using a flow cytometer.

Affinity of antibodies for antigens is generally said to have Kd at alevel of 10 nM to 100 pM. On the other hand, binding strength of theTim-4 protein and phosphatidylserine has been reported to be Kd=about 2nM (Nature (Impact Factor: 41.46). December 2007; 450 (7168): 435-9.DOI: 10.1038/nature 06307). It was surprising that sensitivity of thedetection method of the present invention for detecting theextracellular membrane vesicle or the virus, and the like, using the Timprotein pertaining to the present invention is higher as compared with aconventional method for detecting the extracellular membrane vesicle orthe virus using the antibodies, although affinity of antibodies againstsurface antigens of the extracellular membrane vesicle or the virusenvelope membranes, and their surface antigens is comparable degree toaffinity between phosphatidylserine on the surface of the extracellularmembrane vesicle or the virus envelope membrane and the Tim protein ofthe present invention.

The extracellular membrane vesicle or the virus obtained by the methodof the present invention can be used for analyzing proteins and nucleicacids, such as microRNAs, which are present on the surface or inside ofthe particle, and for basic research of functional analysis of theextracellular membrane vesicle or the virus, or the like. In addition,it can also be utilized for diagnostic drugs, medicines, vaccines, orthe like.

<9. Kit for Capturing Extracellular Membrane Vesicle or the Virus of thePresent Invention>

The Kit for capturing the extracellular membrane vesicle or the virus ofthe present invention comprises, as components:

1. a reagent comprising the Tim protein pertaining to the presentinvention, and a reagent comprising the carrier pertaining to thepresent invention, or 2. a reagent comprising the Tim carrier of thepresent invention.

These kits may further contain at least one kind selected from thecalcium ion-containing washing solution, the protein denaturingagent-containing solution, and the calcium ion chelatingagent-containing solution. Specific examples of each component,preferable aspects, and the like, are as described above.

In addition, in the reagents contained in these kits, there may becontained reagents usually used in this field, for example, the bufferagent, a sensitizer, a surfactant, a preservative (for example, sodiumazide, salicylic acid, benzoic acid, or the like), a stabilizer (forexample, albumin, globulin, water-soluble gelatin, the surfactant,saccharides, or the like), an activator, an effect avoidance agent ofco-present substances, and the one usually used in this field, whichdoes not inhibit stability with co-present reagents, and does notinhibit a reaction or binding of the Tim protein and the carrierpertaining to the present invention, the Tim protein pertaining to thepresent invention bound to the carrier pertaining to the presentinvention and the extracellular membrane vesicle in the sample. Inaddition, concentration ranges, and the like, of these reagents, and thelike, may be used, according to appropriate selection of the usuallyused concentration ranges, and the like, to exert effect each of thereagents has.

Furthermore, the kit of the present invention may contain description ofthe obtaining method of the present invention, the removing method ofthe present invention, the detection method of the present invention, orthe like. The “description” means an instruction manual, a packageinsert, a pamphlet (leaflet), or the like, of the kit, in whichfeatures, principles, operation procedures, determination procedures,and the like, of the method are substantially described by documents orfigures.

Explanation will be given below on the present invention in more detailwith reference to Experimental Examples, Examples and ComparativeExamples, however, the present invention should not be limited at all bythem.

It should be noted that the Tim protein of the present invention, andthe T-cell immunoglobulin and mucin domain-containing molecule-2 (Tim-2)protein (hereinafter it may be abbreviated as “the Tim-2 protein”, insome cases) together may be abbreviated as “the Tim family protein”, insome cases.

EXAMPLES Experimental Example 1. Preparation of Fc Tag Fusion Type Tim-4Protein

The Fc tag fusion type Tim-4 protein was prepared by the followingmethod.

<(1) Vector Construction and Culture Conditions>

First, a vector was constructed for expression of the Fc tag fusion typeTim-4 protein in which a cDNA (SEQ ID NO: 26) encoding the N-terminal 1to 273 amino acid region of a mouse-derived Tim-4 protein wasincorporated into the Sall-EcoRV site of a pEF-Fc vector (hereinafter itmay be abbreviated as “the pEF-Tim-4-Fc”, in some cases).

On the other hand, 293 T cells (RIKEN BRC) were each cultured for 1 dayusing 25 sheets of 150 mm dishes for cell culture, using 20 mL of DMEM(produced by Nacalai Tesque, Inc.) containing 10% FBS (produced byBioWest Co., Ltd.). Thereafter, for each dish, 25 mL of DMEM containing10% FBS was replaced with 25 mL of FBS-free DMEM.

Thereafter, 20 μg of the pEF-Tim-4-Fc was transfected into each of 293 Tcells, using Polyethylenimine “MAX” (produced by Polysciences Inc.),according to a usual method. After transfection, the 293 T cells aftertransfection were each cultured for 4 days under condition at 37° C.,and 5% CO₂.

<(2) Purification>

The resulting culture of the transfected 293T cells was each subjectedto centrifugal separation treatment (800×G, for 5 minutes) to recoverand pool each culture supernatant. The resulting culture supernatant wassubjected to filtration treatment using a Rapid Flow 0.2 μm filter unit(manufactured by Thermo Fisher Scientific Inc.) to separate impuritiesand obtain a culture supernatant filtrate.

Subsequently, 500 mL of the resulting culture supernatant filtrate wasadded to a poly prep column (manufactured by Bio-Rad Laboratories, Inc.)packed with 700 μL of a nProtein A Sepharose 4 Fast Flow (produced by GEHealthcare Japan; Protein A has affinity for the Fc tag) washed with 20mL of PBS, and the Fc tag fusion type Tim-4 protein in the culturesupernatant filtrate was bound on the nProtein A Sepharose 4 Fast Flow.Then, the nProtein A Sepharose 4 Fast Flow was washed with 20 mL of PBS.Thereafter, using each 600 μL of a 0.1 M glycine hydrochloride buffersolution (pH 3.0), elution was carried out 5 times, in 100 μL of a 1 MTris buffer solution (pH 8.0), as a neutralizing solution, to obtaineach 600 μL of five eluate fractions.

Absorbance at 280 nm was measured for each of the resulting five eluatefractions to determine presence or absence of a protein. Fractionscontaining the protein were mixed into one portion, and concentrated byultrafiltration using an Amicon Ultra-0.5 mL, 10K centrifugal filtercolumn (manufactured by Millipore Corp.), and then the solvent wasreplaced with 40 μL of PBS using the same column. Then, amount of theprotein in the eluate replaced with PBS was quantitated by a BCA method,then concentration of the protein was adjusted to 88 μg/mL using PBS toobtain the PBS solution containing the Fc tag fusion type mouse-derivedTim-4 protein (it may be abbreviated as “the Fc tag fusion type mTim-4protein”, in some cases) (the solution may be abbreviated as “the Fc tagfusion type mTim-4 protein-containing PBS solution”, in some cases).

Experimental Example 2. Preparation of FLAG Tag Fusion Type Tim-4Protein

The FLAG tag fusion type Tim-4 protein was prepared by the followingmethod.

<(1) Vector Construction and Culture Conditions>

First, a FLAG tag fusion type mouse-derived Tim-4 protein cDNA (a cDNAencoding an amino acid sequence fused with 1×FLAG at the C-terminal ofthe N-terminal 1 to 273 amino acid region of the mouse-derived Tim-4protein, SEQ ID NO: 33 (containing a stop codon (taa) and a nucleotidesequence encoding 1×FLAG, in this regard, however, excluding therestriction enzyme site), produced by Fasmac Co., Ltd.), wasincorporated in an XhoI/BamHI site of a pCAG-Neo vector (produced byWako Pure Chemical Industries, Ltd.) to construct a vector forexpressing the FLAG tag fusion type Tim-4 protein (hereinafter it may beabbreviated as “the pCAG-Tim-4-FLAG”, in some cases).

On the other hand, the 293 T cells (RIKEN BRC) were cultured for 1 dayusing 50 mL DMEM (produced by Wako Pure Chemical Industries, Ltd.)containing 10% FBS (produced by Biosera) in a 225 cm² flask by seeding,so that the cells grow to 70% to 90% at the time of transfection.Thereafter, 60 μg of the pCAG-Tim-4-FLAG was transfected into the 293 Tcells cultured for 1 day using a Lipofectamine 2000 (produced by ThermoFisher Scientific Inc.), according to a usual method. Aftertransfection, the 293 T cells transfected were cultured for 1 day undercondition at 37° C. and 5% CO₂. Thereafter, the whole amount of theculture solution was removed, the transfected 293 T cells were washedtwice with 10 mL of PBS, and the culture solution was exchanged with 50mL of an Opti-MEM (produced by Thermo Fisher Scientific Inc.) to culturefor 3 days under condition at 37° C. and 5% CO₂.

<(2) Purification>

The culture solution of the 293 T cells transfected, after three days ofculture, was subjected to centrifugal separation treatment (300×G, for 5minutes) to recover the culture supernatant. The recovered culturesupernatant was further subjected to centrifugal separation treatmentthree times (first time: 300×G, for 3 minutes, second time: 1200×G, for20 minutes, and third time: 10000×G, for 20 minutes) to obtain asupernatant, where impurities were separated. The resulting supernatantwas subjected to ultrafiltration using a Vivaspin (cut off molecularweight of 30,000, manufactured by GE Healthcare) to concentrate 10 timesto obtain a concentrated culture supernatant solution. An ANTI-FLAG M2affinity gel (500 μL, 1/10 volume of the concentrated culturesupernatant solution, produced by Sigma-Aldrich Co., Ltd.) washed withPBS was added to 5 mL of the resulting concentrated culture supernatantsolution and mixed with inversion for 3 hours, and the FLAG tag fusiontype Tim-4 protein in the concentrated culture supernatant solution andthe ANTI-FLAG M2 affinity gel were subjected to a reaction to bind theFLAG tag fused Tim-4 protein to the ANTI-FLAG M2 affinity gel. Then, theANTI-FLAG M2 affinity gel was washed three times with 5 μL of PBS.

After that, 250 μL (half volume of the ANTI-FLAG M2 affinity gel used)of a 200 μg/mL FLAG peptide solution (a solution of a DYKDDDDK peptide(produced by Wako Pure Chemical Industries, Ltd.) diluted with PBS) wasadded to the ANTI-FLAG M2 affinity Gel, and mixed with inversion at 4°C. for 30 minutes, and after centrifugal separation treatment (at 4° C.,at 8000×G, for 1 minute), the supernatant (eluate) was recovered. Inorder to remove the FLAG peptide in the recovered supernatant (eluate),20 mL of PBS was added to the supernatant (eluate) and subjected toultrafiltration to obtain a concentrated solution. PBS was added to theresulting concentrated solution, and volume of the solution was adjustedto 500 μL to obtain the PBS solution containing the FLAG tag fusion typemouse-derived Tim-4 protein (it may be abbreviated as “the FLAG tagfusion type mTim-4 protein”, in some cases) (the solution may beabbreviated as “the FLAG tag fusion type mTim-4 protein-containing PBSsolution”, in some cases) (Experimental Example 2).

Experimental Example 3. Preparation of His Tag Fusion Type Tim-4 Protein

The His tag fusion type Tim-4 protein was prepared by the followingmethod.

<(1) Vector Construction and Culture Conditions>

The His tag fusion type mouse-derived Tim-4 protein cDNA (a cDNAencoding an amino acid sequence fused with 6×His tag at the C-terminalof the N-terminal 1 to 273 amino acid region of the mouse-derived Tim-4protein, SEQ ID NO: 34 (containing the stop codon (tga) and a cDNAencoding 6×His tag) was incorporated in the XhoI/BamHI site of thepCAG-Neo vector (produced by Wako Pure Chemical Industries, Ltd.) toconstruct a vector for expressing the His tag fusion type Tim-4 protein(hereinafter it may be abbreviated as “the pCAG-Tim-4-His”, in somecases).

On the other hand, the 293 T cells (RIKEN BRC) were cultured for 1 dayusing 50 mL of DMEM (manufactured by Wako Pure Chemical Industries,Ltd.) containing 10% FBS (produced by Biosera) in a 225 cm² flask.Thereafter, 60 μg of the pCAG-Tim-4-His was transfected into the 293 Tcells using Lipofectamine 2000 (produced by Thermo Fisher ScientificInc.), according to a usual method. After transfection, the 293 T cellstransfected were cultured for 1 day under condition at 37° C. and 5%CO₂. Thereafter, the whole amount of the culture solution was removed,the transfected 293 T cells were washed twice with 10 mL of PBS, and theculture solution was exchanged with 50 mL of the Opti-MEM (manufacturedby Thermo Fisher Scientific Inc.) and cultured for 3 days undercondition at 37° C. and 5% CO₂.

<(2) Purification>

The culture solution of the 293 T cells transfected after three days ofculture was subjected to centrifugal separation treatment (300×G, for 5minutes) to recover the culture supernatant. The recovered culturesupernatant was further subjected to centrifugal separation treatmentthree times (the first time: 300×G, for 3 min, the second time: 1200×G,for 20 min, and the third time: 10000×G, for 20 min) to separateimpurities, and obtain the supernatant. The resulting supernatant wassubjected to ultrafiltration using the Vivaspin (cut off molecularweight 30,000, manufactured by GE Healthcare) and concentrated 10 timesto obtain a concentrated culture supernatant solution.

Ni Sepharose 6 Fast Flow (2.7 mL, produced by GE Healthcare) wasprepared according to a protocol attached to the Ni Sepharose 6 FastFlow, and transferred to a column. A binding buffer (50 mM Tris-HCl, 500mM NaCl, 20 mM imidazole) was added to the column at a flow rate ofabout 1 mL/min, while measuring an OD 280 nm until absorbance at 280 nmwas stabilized. After the addition of 5 mL of the resulting concentratedculture supernatant solution to the column, the binding buffer wassufficiently added and washed until the absorbance was stabilized.Subsequently, an elution buffer (50 mM Tris-HCl, 500 mM NaCl, 300 mMimidazole) was added to the column, and each 1 mL of the eluatefractions was recovered by 10 fractions.

<(3) Electrophoresis and Silver Staining>

Each 15 μL aliquot of the eluted fractions was fractionated and mixedwith 5 μL of 4×sample buffer solution (produced by Wako Pure ChemicalIndustries, Ltd.), and incubated at 98° C. for 5 minutes to obtain each20 μL of a sample for electrophoresis. Each 15 μL of the sample forelectrophoresis was loaded on a Super Sep Ace 5-20% gel (produced byWako Pure Chemical Industries, Ltd.), and subjected to electrophoresisat 25 mA for 65 minutes.

The gel was stained with Silver Stain II Kit Wako (manufactured by WakoPure Chemical Industries, Ltd.) to select and pool (put together) fivefractions containing a target protein. Then, to remove imidazolecontained in the eluate, 20 mL of PBS was added and ultrafiltration wascarried out using the Vivaspin (cut off molecular weight 30,000,produced by GE Healthcare) to obtain a concentrated solution. PBS wasadded to the resulting concentrated solution, and volume of the solutionwas adjusted to 500 μL to obtain the PBS solution containing the His tagfusion type mouse-derived Tim-4 protein (it may be abbreviated as “theHis tag fusion type mTim-4 protein”, in some cases) (the solution may beabbreviated as “the His tag fusion type mTim-4 protein-containing PBSsolution”, in some cases).

Examples 1 to 8. Obtaining of Extracellular Membrane Vesicles UsingTim-4 Carrier of the Present Invention (Obtaining Method of the PresentInvention)

The Tim-4 carrier of the present invention was prepared as follows, andthe extracellular membrane vesicles pertaining to the present inventionwere obtained using this.

<(1) Preparation of Calcium Ion-Containing Culture Supernatant Sample>

A human chronic myelogenous leukemia cell strain K562, 1×10⁷ cells,secreting the extracellular membrane vesicle, was cultured for 3 daysunder condition at 37° C. and 5% CO₂, using 80 mL of the X-VIVO 15medium (produced by Lonza AG). Then the cells were subjected tocentrifugal separation treatment (300×G, for 5 minutes) to precipitatethe cells, and remove the supernatant. The precipitated cells weresuspended in 60 mL of the X-VIVO 15 medium containing 10 μM monensinsodium (produced by MP Biomedicals Co., Ltd.) and cultured for 24 hoursunder condition at 37° C. and 5% CO₂.

Thereafter, the culture solution was subjected to centrifugal separationtreatment (300×G, for 5 minutes) to recover the culture supernatant. Therecovered culture supernatant (60 mL) was further subjected tocentrifugal separation treatment three times (the first time, 300×G, for3 minutes, the second time, 1200×G, for 20 minutes, and the third time,10000×G, for 20 minutes) to separate impurities and obtain thesupernatant. The resulting supernatant was subjected to ultrafiltrationusing the Vivaspin (cut off molecular weight 30,000, produced by GEHealthcare) until volume attained 6 mL or less to obtain theconcentrated solution. The X-VIVO 15 medium containing 10 μM monensinsodium (manufactured by MP Biomedicals Co., Ltd.) was added to theresulting concentrated solution to adjust liquid volume to 6 mL toobtain the 10 times concentrated K562 cell culture concentrated solutionsample (hereinafter it may be abbreviated as “the culture supernatantsample”, in some cases).

In addition, CaCl₂ was added to the resulting culture supernatantsample, so as to attain the final concentration of 2 mM to obtain theK562 cell culture supernatant concentrated sample containing 2 mM CaCl₂(hereinafter it may be abbreviated as “the calcium ion-containingculture supernatant sample”, in some cases).

<(2) Biotin Labeling of Fc Tag Fusion Type Mouse-Derived Tim-4 Protein>

For 114 μL of the PBS solution containing the Fc tag fusion type mTim-4protein (containing 10 μg of the Fc tag fusion type mTim-4 protein)prepared by the same method as in Experimental Example 1, the NH₂-groupof the Fc tag fusion type mTim-4 protein was labeled with biotin, usingthe Biotin Labeling Kit-NH₂ (manufactured by Dojindo MolecularTechnologies, Inc.), according to a protocol attached to the kit toobtain 100 μL of the PBS solution containing 3.8 μg of the NH₂-groupbiotin-labeled Fc tag fusion type mouse-derived Tim-4 protein (it may beabbreviated as “the NH₂-group biotin-labeled Fc tag fusion type mTim-4protein”, in some cases) (the solution may be abbreviated as “theNH₂-group biotin-labeled Fc tag fusion type mTim-4 protein-containingPBS solution”, in some cases).

In addition, for 114 μL of the Fc tag fusion type mTim-4protein-containing PBS solution (containing 10 μg of the Fc tag fusiontype mouse-derived Tim-4 protein) prepared by the same method as inExperimental Example 1, the SH-group of the Fc tag fusion typemouse-derived Tim-4 protein was labeled with biotin, using a BiotinLabeling Kit-SH manufactured by Dojindo Molecular Technologies, Inc.),according to a protocol attached to the kit to obtain 100 μL of the PBSsolution containing 5.9 μg of the SH-group biotin-labeled Fc tag fusiontype mouse-derived Tim-4 protein (it may be abbreviated as “the SH-groupbiotin-labeled Fc tag fusion type mTim-4 protein”, in some cases) (thesolution may be abbreviated as “the SH-group biotin-labeled Fc tagfusion type mTim-4 protein-containing PBS solution”, in some cases).

<(3) Dilution of Fc Tag Fusion Type Mouse-Derived Tim-4Protein-Containing PBS Solution>

The Fc tag fusion type mTim-4 protein-containing PBS solution (11.4 μL),prepared by the same method as in Experimental Example 1, was mixed with188.6 μL of PBS to obtain 200 μL of the PBS solution containing 1 μg ofthe biotin-unlabeled Fc tag fusion type mTim-4 protein.

The NH₂-group biotin-labeled Fc tag fusion type mTim-4protein-containing PBS solution (26.3 μL) prepared above (containing 1μg of the NH₂-group biotin-labeled Fc tag fusion type mTim-4 protein)was mixed with 173.7 μL of PBS to obtain 200 μL of the PBS solutioncontaining 1 μg of the NH₂-group biotin-labeled Fc tag fusion typemTim-4 protein.

In addition, 16.9 μL of the SH-group biotin-labeled Fc tag fusion typemTim-4 protein-containing PBS solution prepared above (containing 1 μgof the SH-group biotin-labeled Fc tag fusion type mTim-4 protein) wasmixed with 183.1 μL of PBS to obtain 200 μL of the PBS solutioncontaining 1 μg of the SH-group biotin-labeled Fc tag fusion type mTim-4protein.

<(4) Washing of Beads>

A PBS-T solution (20 μL) containing a 30 μg/μL Dynabeads Protein G(produced by Thermo Fisher Scientific Inc.) (containing 0.6 mg of theDynabeads Protein G) was dispensed into each of three 1.5 mL tubes(manufactured by BM Equipment Co., Ltd.). Next, 500 μL of PBS was addedto each of the 1.5 mL tubes, and after stirring, each of the 1.5 mLtubes were loaded on a magnet stand, and the Dynabeads Protein G wascollected on the tube wall using magnetic force, and the solution in the1.5 mL tube was discarded with a pipette (hereinafter it may beabbreviated as “the washing operation”, in some cases).

In addition, 60 μL of the PBS solution containing a 10 μg/μL DynabeadsM-270 Streptavidin C1 (produced by Thermo Fisher Scientific Inc.)(containing 0.6 mg of the Dynabeads M-270 Streptavidin C1) was dispensedinto the 1.5 mL tube (manufactured by BM Instruments co., Ltd), and thewashing operation was carried out in the same method as above using 500μL of PBS.

<(5) Immobilization of Fc Tag Fusion Type Mouse-Derived Tim-4 Protein onBeads>

For one of the three 1.5 mL tubes containing the Dynabeads Protein G(0.6 mg), 200 μL of the PBS solution containing 1 μg of thebiotin-unlabeled Fc tag fusion type mTim-4 protein prepared above wasadded in its entirety, and subjected to a reaction at 8° C. for 1 hourto obtain 200 μL of the PBS solution containing the carrier on which thebiotin-unlabeled Fc tag fusion type mTim-4 protein was bound (mTim-4carrier).

For one of the remaining two 1.5 mL tubes containing the DynabeadsProtein G (0.6 mg), 200 μL of the PBS solution containing 1 μg of theNH₂-group biotin-labeled Fc tag fusion type mTim-4 protein preparedabove was added in its entirety, and subjected to a reaction at 8° C.for 1 hour to obtain 200 μL of the PBS solution containing the carrieron which the NH₂-group biotin-labeled Fc tag fusion type mTim-4 proteinwas bound (mTim-4 carrier).

In addition, for one tube, 200 μL of the PBS solution containing 1 μg ofthe SH-group biotin-labeled Fc tag fusion type mTim-4 protein preparedabove was added in its entirety, and subjected to a reaction at 8° C.for 1 hour to obtain 200 μL of the PBS solution containing the carrieron which the SH-group biotin-labeled Fc tag fusion type mTim-4 proteinwas bound (mTim-4 carrier).

For one of 1.5 mL tube containing the Dynabeads M-270 Streptavidin C1,200 μL of the PBS solution containing 1 μg of the SH-groupbiotin-labeled Fc tag fusion type mTim-4 protein prepared by the samemethod as above was added, and subjected to a reaction at 8° C. for 1hour to obtain 200 μL of the PBS solution containing the carrier onwhich the SH-group biotin-labeled Fc tag fusion type mTim-4 protein wasbound (mTim-4 carrier).

In this way, 200 μL of the PBS solution containing 0.6 mg of each of thefour types of the mTim-4 carriers shown in the following Table 1 wasobtained.

TABLE 1 1 2 3 4 Mouse- Fc tag fusion Unlabeled Biotin labeled Biotinlabeled derived type mouse- (NH₂-group) (SH-group) Tim-4 derived Tim-4carrier protein Carrier Protein G Streptavidin beads beads<(6) Obtaining of Extracellular Membrane Vesicles by Obtaining Method ofthe Present Invention>

The resulting 0.6 mg of the four types of the mTim-4 carriers above,described in Table 1, was subjected to the washing operation each threetimes with 500 μL of PBS, and then 200 μL of the calcium ion-containingculture supernatant sample prepared in the (1) was added to each of themTim-4 carriers in a pellet state and subjected them to a reaction at 8°C. for 3 hours.

The mTim-4 carrier after the reaction was subjected to the washingoperation 3 times with 500 μL each of TBS-T containing 2 mM CaCl₂ (Trisbuffer, 0.05% Tween 20, 2 mM CaCl₂). At the third time of the washingoperation, each 250 μL of the 4 types of the mTim-4 carriers wasdispensed into two 1.5 mL tubes.

Each 20 μL of the 1% SDS aqueous solution or the 1 mM EDTA aqueoussolution was added, as the eluent, to each 0.3 mg of the four types ofthe mTim-4 carriers in a pellet state, and then they were mixed at roomtemperature for 10 seconds using a vortex mixer and spun down. Each 1.5mL tube was loaded on a magnet stand, and the mTim-4 carrier wascollected on the tube wall using magnetic force to recover thesupernatant (eluate).

It should be noted that, the types of the mTim-4 proteins, the carriersand the eluents used to obtain the extracellular membrane vesicles fromthe mTim-4 carrier, used in each Example, and the lane numbers inWestern blotting to be described later are shown in the following Table2.

TABLE 2 Example 1 Example 2 Example 3 Example 4 Example 5 Example 6Example 7 Example 8 Mouse- Fc tag fusion Unlabeled Biotin labeled Biotinlabeled derived type mouse- (NH₂-group) (SH-group) Tim-4 derived carrierTim-4 protein Carrier Protein G beads Streptavidin beads Eluent 1% 1 mM1% 1 mM 1% 1 mM 1% 1 mM SDS EDTA SDS EDTA SDS EDTA SDS EDTA Lane numberin FIG. 1 1 2 3 4 5 6 7 8<(7) Western Blotting>

To each 7.5 μL of the supernatants (eluates) obtained in Examples 1 to8, 2.5 μL of the 4×sample buffer (produced by Wako Pure ChemicalIndustries, Ltd.) was added and incubated at 98° C. for 5 minutes toobtain each sample for Western blotting. Each 10 μL of the sample forWestern blotting was loaded on the Super Sep Ace 5-20% gel (produced byWako Pure Chemical Industries, Ltd.), and subjected to electrophoresisat 25 mA for 65 minutes. The resulting electrophoresis gel wastranscribed onto a PVDF membrane (produced by Millipore Corporation)under 1 mA/cm² for 60 minutes using a semi-dry blotter and adiscontinuous buffer (Anode buffer 1: 0.3 M Tris/20% methanol, Anodebuffer 2: 0.025 M Tris/20% methanol, Cathode buffer: 0.025 M Tris/0.04 Maminocaproic acid/20% methanol).

To the PVDF membrane, 3% skimmed milk diluted with PBS-T (the PBSbuffer, 0.1% Tween 20) was added and subjected to a reaction at roomtemperature for 1 hour for blocking, and 2 mL of an anti-human Lamp-1mouse monoclonal antibody diluted 250-fold with PBS-T (produced by BDBiosciences, hereinafter it may be abbreviated as “the anti-human Lamp-1antibody”, in some cases) was subjected to a reaction at 8° C.overnight.

After that, the PVDF membrane after the reaction was washed three timeswith PBS-T, and then subjected to a reaction with a secondary antibody{anti-mouse IgG (H+L), rabbit, IgG fraction, peroxidase-conjugatedantibody} (produced by Wako Pure Chemical Industries, Ltd.) diluted10,000-fold with PBS-T at room temperature for 1 hour. After washing it5 times with PBS-T, ImmunoStar Zeta (produced by Wako Pure ChemicalIndustries, Ltd.) was added to detect a luminescent signal usingLAS-4000 (manufactured by General Electric Company). It should be notedthat the anti-human Lamp-1 antibody is an antibody against Lamp-1 whichis one of the marker proteins of exosome.

<Results>

The results of Western blotting obtained are shown in FIG. 1. In FIG. 1,each lane is as follows:

Lane 1: Results of Example 1 (results of the case of using the mTim-4carrier in which the biotin-unlabeled Fc tag fusion type mTim-4 proteinwas bound on the Protein G beads, and using the 1% SDS aqueous solutionas the eluent);

Lane 2: Results of Example 2 (the results of the case of using themTim-4 carrier in which the biotin-unlabeled Fc tag fusion type mTim-4protein was bound on the Protein G beads, and using the 1 mM EDTAsolution as the eluent);

Lane 3: Results of Example 3 (the results of the case of using themTim-4 carrier in which the NH₂-group biotin-labeled Fc tag fusion typemTim-4 protein was bound on the Protein G beads, and using the 1% SDSaqueous solution as the eluent);

Lane 4: Results of Example 4 (the results of the case of using themTim-4 carrier in which the NH₂-group biotin-labeled Fc tag fusion typemTim-4 protein was bound on the Protein G beads, and using the 1 mM EDTAsolution as the eluent);

Lane 5: Results of Example 5 (the results of the case of using themTim-4 carrier in which the SH-group biotin-labeled Fc tag fusion typemTim-4 protein was bound on the Protein G beads, and using the 1% SDSaqueous solution as the eluent);

Lane 6: Results of Example 6 (the results of the case of using themTim-4 carrier in which the SH-group biotin-labeled Fc tag fusion typemTim-4 protein was bound on the Protein G beads, and using the 1 mM EDTAsolution as the eluent);

Lane 7: Results of Example 7 (the results of the case of using themTim-4 carrier in which the SH-group biotin-labeled Fc tag fusion typemTim-4 protein was bound to the streptavidin beads, and using the 1% SDSaqueous solution as the eluent);

Lane 8: Results of Example 8 (the results of the case of using themTim-4 carrier in which the SH-group biotin-labeled Fc tag fusion typemTim-4 protein was bound to the streptavidin beads, and using the 1 mMEDTA solution as the eluent).

From FIG. 1, it has been revealed that the extracellular membranevesicles including exosome can be obtained, according to the obtainingmethod of the present invention, from the fact that a band of Lamp-1,which is an exosome marker, was recognized at the vicinity of 100 kDa inany of Examples 1 to 8

Examples 1 to 8: The Lanes 1 to 8

In particular, from comparison among Examples 2, 4, 6, and 8, it hasbeen revealed that, when the calcium ion chelating agent is used as theeluent, the one in which the Tim-4 protein and the carrier are bound viathe SH-group of the Tim-4 protein (Example 8: the lane 8) is possible toobtain more extracellular membrane vesicles, as compared with the onebound via the NH₂-group (Example 4: the lane 4) or those bound via theaffinity tag (Example 2: lane 2, Example 6: the lane 6) (Example 2: lane2, Example 4: lane 4, Example 6: lane 6, Example 8: the lane 8).

Examples 9 to 16. Obtaining of Extracellular Membrane Vesicles UsingTim-4 Carrier of the Present Invention (Obtaining Method of the PresentInvention)

The Tim-4 carrier of the present invention was prepared as follows, andthe extracellular membrane vesicles pertaining to the present inventionwere obtained using the same.

<(1) Preparation of Culture Supernatant Sample>

Preparation of the culture supernatant sample was carried out in thesame method as in “(1) preparation of culture supernatant sample” ofExamples 1 to 8.

<(2) Biotin Labeling of Fc Tag Fusion Type Mouse-Derived Tim-4 Protein>

For 114 μL of the PBS solution containing the Fc tag fusion type mTim-4protein (containing 10 μg of the Fc tag fusion type mTim-4 protein),prepared by the same method as in “(2) Biotin labeling of Fc tag fusiontype mouse-derived Tim-4 protein” of Experimental Example 1, theSH-group of the Fc tag fusion type mTim-4 protein was labeled withbiotin by the same method as in Example 1 to obtain 100 μL of the PBSsolution containing 3.9 μg of the SH-group biotin-labeled Fc tag fusiontype mTim-4 protein (hereinafter it may be abbreviated as “the SH-groupbiotin-labeled Fc tag fusion type mTim-4 protein-containing PBSsolution”, in some cases).

<(3) Biotin Labeling of FLAG Tag Fusion Type Mouse-Derived Tim-4Protein>

For 99 μL of the PBS solution containing the FLAG tag fusion type mTim-4protein prepared by the same method as in Experimental Example 2(containing 10 μg of the FLAG tag fusion type mTim-4 protein), theSH-group of the FLAG tag fusion type mTim-4 protein was labeled withbiotin using the Biotin Labeling Kit-SH (manufactured by DojindoMolecular Technologies, Inc.), according to a protocol attached to thekit to obtain 100 μL of the PBS solution containing 4.6 μg of theSH-group biotin-labeled FLAG tag fusion type mTim-4 protein (hereinafterit may be abbreviated as “the PBS solution containing the SH-groupbiotin-labeled FLAG tag fusion type mTim-4 protein”, in some cases).

<(4) Biotin Labeling of his Tag Fusion Type Mouse-Derived Tim-4 Protein>

For 54 μL of the PBS solution containing the His tag fusion type mTim-4protein prepared by the same method as in Experiment Example 3(containing 10 μg of the His tag fusion type mTim-4 protein), theSH-group of the His tag fusion type mTim-4 protein was labeled withbiotin using the Biotin Labeling Kit-SH (manufactured by DojindoMolecular Technologies, Inc.), according to a protocol attached to thekit to obtain 100 μL of the PBS solution containing 7.2 μg of theSH-group biotin-labeled His tag fusion type mTim-4 protein (hereinafterit may be abbreviated as “the PBS solution containing the SH-groupbiotin-labeled His tag fusion type mTim-4 protein”, in some cases).

<(5) Dilution of Tag Fusion Type Mouse-Derived Tim-4 Protein>

The Fc tag fusion type mTim-4 protein-containing PBS solution (11.4 μL),prepared by the same method as in Experimental Example 1 (containing 1μg of the Fc tag fusion type mouse-derived Tim-4 protein), was mixedwith 188.6 μL of PBS to obtain 200 μL of the PBS solution containing 1μg of biotin-unlabeled Fc tag fusion type mouse-derived Tim-4 protein.

The SH-group biotin-labeled Fc tag fusion type mTim-4 protein-containingPBS solution (16.9 μL), prepared in the (2) (containing 1 μg of theSH-group biotin-labeled Fc tag fusion type mTim-4 protein), was mixedwith 183.1 μL of PBS to obtain 200 μL of the PBS solution containing 1μg of the SH-group biotin-labeled Fc tag fusion type mTim-4 protein.

In addition, 21.6 μL of the SH-group biotin-labeled FLAG tag fusion typemTim-4 protein-containing PBS solution, prepared in the (3) (containing1 μg of the SH-group biotin-labeled FLAG tag fusion type mTim-4protein), was mixed with 178.4 μL of PBS to obtain 200 μL of the PBSsolution containing 1 μg of the SH-group biotin-labeled FLAG tag fusiontype mTim-4 protein.

The SH-group biotin-labeled His tag fusion type mTim-4protein-containing PBS solution (13.9 μL), prepared in the (4)(containing 1 μg of the SH-group biotin-labeled His tag fusion typemTim-4 protein), was mixed with 186.1 μL of PBS to obtain 200 μL of thePBS solution containing 1 μg of the SH-group biotin-labeled His tagfusion type mTim-4 protein.

<(6) Washing of Beads>

The PBS-T solution (20 μL) containing 30 μg/μL of the Dynabeads ProteinG (produced by Thermo Fisher Scientific, Inc.) (containing 0.6 mg of theDynabeads Protein G) was dispensed into one 1.5 mL tube (manufactured byBM Equipment Co., Ltd.), and the washing operation was carried out using500 μL of PBS in the same method as in “(4) washing of beads” inExamples 1 to 8.

In addition, 60 μL of the PBS solution containing the 10 μg/μL DynabeadsM-270 Streptavidin (produced by Thermo Fisher Scientific Inc.)(containing 0.6 mg of the Dynabeads M-270 Streptavidin) was dispensedinto each of the three 1.5 mL tubes (manufactured by BM Equipment Co.,Ltd.), and the washing operation was carried out using each 500 μL ofPBS in the same method as in “(4) washing of beads” in Examples 1 to 8.

<(7) Immobilization of Tag-Fused Mouse-Derived Tim-4 Protein on Beads>

Subsequently, 200 μL of the PBS solution containing 1 μg of thebiotin-unlabeled Fc tag fusion type mTim-4 protein was added in itsentirety to the 1.5 mL tube containing 0.6 mg of the Dynabeads Protein Gin a pellet state, after the washing operation, and subjected to areaction at 8° C. for 1 hour to obtain 200 μL of the PBS solutioncontaining the carrier on which the biotin-unlabeled Fc tag fusion typemTim-4 protein was bound (the mTim-4 carrier).

Furthermore, for one of the three 1.5 mL tubes containing 0.6 mg of theDynabeads M-270 Streptavidin in a pellet state, after the washingoperation, 200 μL of the PBS solution containing 1 μg of the SH-groupbiotin-labeled Fc tag fusion type mTim-4 protein prepared above wasadded in its entirety and subjected to a reaction at 8° C. for 1 hour toobtain 200 μL of the PBS solution containing the carrier on which theSH-group biotin-labeled Fc tag fusion type mTim-4 protein was bound (themTim-4 carrier).

For one of the remaining two 1.5 mL tubes containing 0.6 mg of theDynabeads M-270 Streptavidin in a pellet state, after the washingoperation, 200 μL of the PBS solution containing 1 μg of the SH-groupbiotin-labeled FLAG tag fusion type mTim-4 protein prepared above wasadded in its entirety, and subjected to a reaction at 8° C. for 1 hourto obtain 200 μL of the PBS solution containing the carrier on which theSH-group biotin-labeled FLAG tag fusion type mTim-4 protein was bound(the mTim-4 carrier).

In addition, for one tube, 200 μL of the PBS solution containing 1 μg ofthe SH-group biotin-labeled His tag fusion type mTim-4 protein preparedabove was added in its entirety, and subjected to a reaction at 8° C.for 1 hour to obtain 200 μL of the PBS solution containing the carrieron which the SH-group biotin-labeled His tag fusion type mTim-4 proteinwas bound (the mTim-4 carrier).

In this way, 200 μL of the PBS solutions containing 0.6 mg of each ofthe four types of mTim-4 carriers shown in the following Table 3 wereobtained.

TABLE 3 1 2 3 4 Mouse-derived Type of tag Fc tag FLAG tag His tag Tim-4carrier Tag fusion type Unlabeled Biotin labeled mouse-derived(SH-group) Tim-4 protein Carrier Protein G Streptavidin beads beads<(8) Obtaining of Extracellular Membrane Vesicles by Obtaining Method ofthe Present Invention>

Each supernatant (eluate) was obtained by carrying out the same methodas in “(6) Obtaining of extracellular membrane vesicles by obtainingmethod of the present invention” of Examples 1 to 8, except for using“0.6 mg of the four kinds of the mTim-4 carriers shown in Table 3”instead of “0.6 mg of the four kinds of the mTim-4 carrier shown inTable 1”. It should be noted that the type of the mTim-4 proteins, thecarriers, and the eluents used to obtain the extracellular membranevesicles from the mTim-4 carrier, used in each Example, and the lanenumbers in Western blotting to be described later are shown in thefollowing Table 4.

TABLE 4 Example 9 Example 10 Example 11 Example 12 Example 13 Example 14Example 15 Example 16 Mouse- Type of tag Fc tag FLAG tag His tag derivedTag fusion Unlabeled Biotin labeled Tim-4 type mouse- (SH-group) carrierderived Tim-4 protein Carrier Protein G beads Streptavidin beads Eluent1% 1 mM 1% 1 mM 1% 1 mM 1% 1 mM SDS EDTA SDS EDTA SDS EDTA SDS EDTA Lanenumber in FIG. 2 1 2 3 4 5 6 7 8<(7) Western Blotting>

Western blotting was carried out by the same method as in “(7) Westernblotting” of Examples 1 to 8, except for using “7.5 μL of eachsupernatant (eluate) obtained in Examples 9 to 16 (above (8)), insteadof “7.5 μL of each supernatant (eluate) obtained in Examples 1 to 8”.

<Results>

The results of Western blotting obtained are shown in FIG. 2. In FIG. 2,each lane is as follows:

Lane 1: Results of Example 9 (the results of the case of using themTim-4 carrier in which the biotin-unlabeled Fc tag fusion type mTim-4protein was bound on the Protein G beads, and using the 1% SDS aqueoussolution as the eluent);

Lane 2: Result of Example 10 (the results of the case of using themTim-4 carrier in which the biotin-unlabeled Fc tag fusion type mTim-4protein was bound on the Protein G beads, and using the 1 mM EDTAsolution as the eluent);

Lane 3: Results of Example 11 (the results of the case of using themTim-4 carrier in which the SH-group biotin-labeled Fc tag fusion typemTim-4 protein was bound on the streptavidin beads, and using the 1% SDSaqueous solution as the eluent);

Lane 4: Results of Example 12 (the results of the case of using themTim-4 carrier in which the SH-group biotin-labeled Fc tag fusion typemTim-4 protein was bound on the streptavidin beads, and using the 1 mMEDTA solution as the eluent);

Lane 5: Results of Example 13 (the results of the case of using themTim-4 carrier in which the SH-group biotin-labeled FLAG tag fusion typemTim-4 protein was bound on the streptavidin beads, and using the 1% SDSaqueous solution as the eluent);

Lane 6: Results of Example 14 (the results of the case of using themTim-4 carrier in which the SH-group biotin-labeled FLAG tag fusion typemTim-4 protein was bound on the streptavidin beads, and using the 1 mMEDTA solution as the eluent);

Lane 7: Results of Example 15 (the results of the case of using themTim-4 carrier in which the SH-group biotin-labeled His tag fusion typemTim-4 protein was bound on the streptavidin beads, and using the 1% SDSaqueous solution as the eluent);

Lane 8: The results of Example 16 (the results of the case of using themTim-4 carrier in which the SH-group biotin-labeled His tag fusion typemTim-4 protein was bound on the streptavidin beads, and using the 1 mMEDTA solution as the eluent).

From FIG. 2, it has been revealed that the extracellular membranevesicles including exosome can be obtained, by using the obtainingmethod of the present invention, because of the fact that a band ofLamp-1, which is a marker protein of exosome, was detected at thevicinity of 100 kDa, in any of Examples 9 to 16 (Example 9 to 16: thelanes 1 to 8).

In addition, it has been revealed that the extracellular membranevesicles can be obtained by using the Tim-4 carrier of the presentinvention, irrespective of the type, length, and presence or absence ofthe tags (Examples 9 to 16: the lanes 1 to 8).

From comparison among Examples 10, 12, 14, and 16, it has been revealedthat when EDTA, which is the calcium ion chelating agent, is used as theeluent, those on which the Tim-4 protein and the carrier are bound viathe SH-group of the Tim-4 protein (Example 12: the lane 4, Example 14:the lane 6, Example 16: the lane 8) are possible to obtain moreextracellular membrane vesicles as compared with the one bound via theaffinity tag (Example 10: the lane 2).

Examples 17 to 18. Obtaining of Extracellular Membrane Vesicles UsingTim-4 Carrier of the Present Invention (Obtaining Method of the PresentInvention)

The Tim-4 carrier of the present invention was prepared as follows, andthe extracellular membrane vesicles pertaining to the present inventionwere obtained using the same.

<(1) Preparation of Culture Supernatant Sample>

Preparation of culture supernatant sample was carried out in the samemethod as in “(1) Preparation of culture supernatant sample” of Examples1 to 8.

<(2) Dilution of Biotin-Unlabeled FLAG Tag Fusion Type Mouse-DerivedTim-4 Protein>

The PBS solution (47 μL) containing 5 μg of the biotin-unlabeled FLAGtag fusion type mTim-4 protein, prepared by the same method as inExperimental Example 2 (the biotin-unlabeled FLAG tag fusion type mTim-4protein-containing PBS solution) was mixed with 153 μL of PBS to obtain200 μL of the PBS solution containing 5 μg of the biotin-unlabeled FLAGtag fusion type mTim-4 protein.

<(3) Washing of Beads>

A 50% glycerol TBS solution (50 μL) containing 10 μg/μL of anti-DYKDDDDKtag antibody magnetic beads (containing 0.5 mg of the anti-DYKDDDDK tagantibody magnetic beads) (produced by Wako Pure Chemical Industries,Ltd.) was dispensed into the 1.5 mL tube (manufactured by BM EquipmentCo., Ltd.), and the washing operation was carried out using 500 μL ofPBS.

<(4) Immobilization of FLAG Tag Fusion Type Mouse-Derived Tim-4 Proteinon Carrier>

Subsequently, 200 μL of the PBS solution containing 5 μg of the FLAG tagfusion type mTim-4 protein was added in its entirety to theanti-DYKDDDDK tag antibody magnetic beads in a pelleted state, after thewashing operation, and subjected to a reaction at 8° C. for 1 hour toobtain 200 μL of the PBS solution containing the mTim-4 carrier.

In this way, 200 μL of the PBS solution containing 0.5 mg of the mTim-4carrier shown in the following Table 5 was obtained.

TABLE 5 1 Mouse-derived Type of tag FLAG tag Tim-4 carrier Tag fusiontype Biotin-unlabeled mouse-derived Tim-4 protein Carrier Anti-DYKDDDDKtag antibody beads<(5) Obtaining of Extracellular Membrane Vesicles by Obtaining Method ofthe Present Invention>

Each supernatant (eluate) was obtained by carrying out the same methodas in “(6) Obtaining of extracellular membrane vesicles by the obtainingmethod of the present invention” of Examples 1 to 8, except for using“0.5 mg of the mTim-4 carrier described in Table 5”, instead of “0.6 mgof the four kinds of the mTim-4 carrier described in Table 1”.

It should be noted that the types of the mTim-4 protein, the carrier andthe eluent used to obtain the extracellular membrane vesicles from themTim-4 carrier, used in each Example, and the lane numbers in Westernblotting to be described later are shown in the following Table 6.

TABLE 6 Example 17 Example 18 Mouse-derived Type of tag FLAG tag Tim-4carrier Tag fusion type Biotin-unlabeled mouse-derived Tim-4 proteinCarrier Anti-DYKDDDDK tag antibody beads Eluent 1% 1 mM SDS EDTA Lanenumber in FIG. 3 1 2<(6) Western Blotting>

Western blotting was carried out by the same method as in “(7) Westernblotting” of Examples 1 to 8, except for using “7.5 μL of eachsupernatant (eluate) obtained in Examples 17 to 18 (above (5)) insteadof “7.5 μL of each supernatant (eluate) obtained in Examples 1 to 8”.

<Results>

The results of Western blotting obtained are shown in FIG. 3. In FIG. 3,each lane is as follows:

Lane 1: Results of Example 17 (the results of the case of using themTim-4 carrier in which the biotin-unlabeled FLAG tag fusion type mTim-4protein was bound on the anti-DYKDDDDK antibody beads, and using the 1%SDS aqueous solution as the eluent);

Lane 2: Result of Example 18 (the results of the case of using themTim-4 carrier in which the biotin-unlabeled FLAG tag fusion type mTim-4protein was bound on the anti-DYKDDDDK antibody beads, and using the 1mM EDTA solution as the eluent).

From FIG. 3, it has been revealed that the extracellular membranevesicles including exosome can be obtained, according to the obtainingmethod of the present invention, when the carrier on which the Tim-4protein is immobilized via the anti-tag antibody, from the fact that aband of Lamp-1, which is an exosome marker, was observed at the vicinityof 100 kDa, in any case of Examples 17 to 18 (Examples 17 to 18: thelanes 1 to 2).

Examples 19 to 20 and Comparative Examples 1 to 3. Comparison of Purityof Extracellular Membrane Vesicles Obtained by the Obtaining Method ofthe Present Invention and Conventional Method

As described below, comparison was carried out on purity of theextracellular membrane vesicles obtained by each of the obtaining methodof the present invention by SDS elution (Example 19), the obtainingmethod of the present invention by EDTA elution (Example 20), theultracentrifugal separation method (Comparative Example 1), Exo Quick(Comparative Example 2), and Total Exosome Isolation (ComparativeExample 3).

<Obtaining of Extracellular Membrane Vesicles by the Obtaining Method ofthe Present Invention (Examples 19 and 20)>

The mTim-4 protein where the SH-group biotin-labeled FLAG tag fusiontype mTim-4 protein was bound to the Dynabeads M-270 Streptavidin C1(produced by Thermo Fisher Scientific Inc.) and the extracellularmembrane vesicles in the calcium ion-containing culture supernatantsample were subjected to a reaction by the same method as in Examples 13to 14, and the extracellular membrane vesicles were each eluted with the1% SDS aqueous solution or 1 mM EDTA aqueous solution, as the eluent, toobtain each of the supernatants (eluates) except for using “500 μL ofthe calcium ion-containing culture supernatant sample”, instead of “200μL of the calcium ion-containing culture supernatant sample”, “50 μL ofthe 1% SDS aqueous solution”, instead of “20 μL of the 1% SDS aqueoussolution” as an eluent, and using “50 μL of the 1 mM EDTA aqueoussolution”, instead of “20 μL of the 1 mM EDTA aqueous solution.

Each of the resulting supernatants (eluates) was defined as Sample 1(when eluted with the 1% SDS aqueous solution), and Sample 2 (wheneluted with the 1 mM EDTA aqueous solution).

It should be noted that the type of the mTim-4 protein, the carrier andthe eluents used to obtain the extracellular membrane vesicles from themTim-4 carrier, used in Examples 19 and 20, are shown in the followingTable 7.

TABLE 7 Example 19 Example 20 Mouse-derived Type of tag FLAG tag Tim-4carrier Tag fusion type Biotin-labeled mouse-derived (SH-group) Tim-4protein Carrier Streptavidin beads Eluent 1% 1 mM SDS EDTA

Obtaining of Extracellular Membrane Vesicles by UltracentrifugalSeparation Method (Comparative Example 1)

The culture supernatant sample (1 mL), prepared by the same method as inExample 1, was subjected to centrifugal separation treatment (20,000×G,for 30 minutes) to separate impurities and obtain the supernatant. Then,1 mL of the resulting supernatant was subjected to ultracentrifugalseparation treatment (110,000×G, for 70 minutes) to obtain a precipitatefraction. Thereafter, the resulting precipitate fraction was suspendedin 1 mL of PBS. The suspension of the precipitate fraction was subjectedto ultracentrifugal separation treatment again (110,000×G, for 70minutes), and then the resulting precipitate fraction was suspended in50 μL of PBS. The resulting suspension was referred to as Sample 3(Comparative Example 1).

Obtaining of Extracellular Membrane Vesicles by Centrifugal SeparationMethod Using Commercially Available Reagent (Obtaining of ExtracellularMembrane Vesicles by ExoQuick) (Comparative Example 2)

The culture supernatant sample (1 mL), prepared by the same method as inExample 1, was subjected to centrifugal separation treatment (20,000×G,for 30 minutes) to separate impurities and obtain the supernatant. Then,1 mL of the resulting supernatant was mixed with 0.2 mL of theExoQuick-TC Reagent (produced by System Biosciences, Inc.) and themixture solution was allowed to stand overnight at 8° C. Thereafter, themixture solution that had been allowed to stand overnight was subjectedto centrifugal separation treatment (1,500×G, for 30 minutes), and theresulting precipitate fraction was suspended in 100 μL of PBS. Theresulting suspension solution was referred to as Sample 4 (ComparativeExample 2).

Obtaining of Extracellular Membrane Vesicles by Centrifugal SeparationMethod Using Commercially Available Reagent (Obtaining of ExtracellularMembrane Vesicles by Total Exosome Isolation) (Comparative Example 3)

The K562 cell culture supernatant concentrated solution sample (1 mL),prepared by the same method as in Example 1, was subjected tocentrifugal separation treatment (20,000×G, for 30 minutes) to separateimpurities and obtain the supernatant. Subsequently, 1 mL of theresulting supernatant was mixed with 0.5 mL of the Total ExosomeIsolation Reagent (produced by Thermo Fisher Scientific Inc.) andallowed to stand at 8° C. for 1 day. The precipitate fraction obtainedby centrifugal separation treatment (10,000×G, for 1 hour) of the mixedsolution was suspended in 100 μL of PBS. The resulting suspensionsolution was referred to as Sample 5 (Comparative Example 3).

It should be noted that the methods used in each Example and ComparativeExample, the resulting samples, and the lane numbers in Western blottingand silver staining to be described later are shown in the followingTable 8.

TABLE 8 Comparative Comparative Comparative Example 19 Example 20Example 1 Example 2 Example 3 Method Method of the Method of the Ultra-Exo Quick Total Exosome present invention present inventioncentrifugation Isolation (elution by (elution by method 1% SDS) 1 mMEDTA) Sample number Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Lanenumber 1 2 3 4 5 in FIG. 4<Western Blotting>

Amounts of the proteins in the resulting Samples 1 to 5 by each methodwere measured by the BCA method, and based on the measurement result,and using the amounts of the proteins as standard amounts ofelectrophoresis, Western blotting was carried out. That is, 37.5 μL ofthe PBS solution containing 0.25 μg of each of the proteins in Samples 1to 5, and 12.5 μL of the 4×sample buffer solution (produced by Wako PureChemical Industries, Ltd.) were mixed, and incubated at 98° C. for 5minutes to obtain 50 μL of each sample for Western blotting.

Subsequently, each 20 μL of the resulting sample for Western blottingwas loaded on two sheets of the Super Sep Ace 5-20% gel (produced byWako Pure Chemical Industries, Ltd.), and subjected to electrophoresisat 25 mA for 60 minutes. One of the resulting two sheets of the gels wastranscribed onto the PVDF membrane (produced by Millipore Corporation)at 1 mA/cm² for 60 minutes, using the semi-dry blotter and thediscontinuous buffer (Anode Buffer 1: 0.3 M Tris/20% Methanol, AnodeBuffer 2: 0.025 M Tris/20% Methanol, Cathode Buffer: 0.025 M Tris/0.04 Maminocaproic acid/20% methanol). The 3% skimmed milk, diluted withPBS-T, was added to the PVDF membrane and subjected to a reaction atroom temperature for 1 hour for blocking, and then 2 mL of theanti-human Lamp-1 mouse monoclonal antibody (produced by BDBiosciences), diluted 250-fold with PBS-T, was subjected to a reactionat room temperature for 1 hour. After washing 3 times with PBS-T, themembrane was subjected to a reaction with the secondary antibody{anti-mouse IgG (H+L), rabbit, the IgG fraction, theperoxidase-conjugated antibody (produced by Wako Pure ChemicalIndustries, Ltd.)}, diluted 10,000-fold with PBS-T, at room temperaturefor 1 hour. After washing five times with PBS-T, an ECL prime (producedby General Electric Company) was added, and each luminescent signal wasdetected using the LAS-4000 (manufactured by General Electric Company).

In addition, the other one gel was silver stained using a Silverstaining II kit Wako (manufactured by Wako Pure Chemical Industries,Ltd.).

<Results>

The results of Western blotting obtained, and the results of silverstaining are shown in FIG. 4-A and FIG. 4-B, respectively. In FIG. 4-Aand FIG. 4-B, each lane is as follows:

Lane 1: Results of Example 19 (the results of the case where the methodof the present invention was carried out using the mTim-4 carrier wherethe SH-group biotin-labeled FLAG tag fusion type mTim-4 protein wasbound on the streptavidin beads, and using the 1% SDS aqueous solutionas the eluent);

Lane 2: Results of Example 20 (the results of the case where the methodof the present invention was carried out using the mTim-4 carrier wherethe SH-group biotin-labeled FLAG tag fusion type mTim-4 protein wasbound on the streptavidin beads, and using the 1 mM EDTA solution as theeluent);

Lane 3: Results of Comparative Example 1 (the results of the case whereobtaining of the extracellular membrane vesicles was carried out by theultracentrifugal separation method);

Lane 4: Results of Comparative Example 2 (the results of the case whereobtaining of the extracellular membrane vesicles was carried out byExoQuick);

Lane 5: Results of Comparative Example 3 (the results of the case whereobtaining of the extracellular membrane vesicles was carried out byTotal Exosome Isolation).

From FIG. 4-A, it has been revealed that the extracellular membranevesicles including exosome can be obtained, according to the obtainingmethod of the present invention, from the fact that a band of Lamp-1,which is an exosome marker, was observed at the vicinity of 100 kDa inany of Examples 19 and 20, which are the obtaining method of the presentinvention (Examples 19 to 20: the lanes 1 to 2).

On the other hand, from FIG. 4-A, a band of Lamp-1, which is an exosomemarker, was only slightly observed in any of the conventional methods(the ultracentrifugal separation method (Comparative Example 1), ExoQuick (Comparative Example 2) and Total Exosome Isolation (ComparativeExample 3)) (Comparative Examples 1 to 3: the lanes 3 to 5).

In addition, from FIG. 4-B, the bands derived from contaminatingproteins and the like were found fewer in the obtaining method of thepresent invention (Examples 19 and 20), as compared with in any of theconventional methods (ultracentrifugal separation method (ComparativeExample 1), Exo Quick (Comparative Example 2), and Total ExosomeIsolation (Comparative Example 3)), (Examples 19 and 20: lanes 1 and 2,Comparative Examples 1 to 3: lanes 3 to 5).

From the above results, it has been revealed that the extracellularmembrane vesicles can be obtained in good purity, by the obtainingmethod of the present invention (Examples 19 and 20), as compared withany of the conventional methods (the ultracentrifugal separation method(Comparative Example 1), Exo Quick (Comparative Example 2), and TotalExosome Isolation 3)) (the lanes 1 to 5).

Example 21. Electron Microscopic Observation of Extracellular MembraneVesicles Obtained by Obtaining Method of the Present Invention

In order to investigate states of the extracellular membrane vesiclesobtained by the obtaining method of the present invention, observationwas carried out with an electron microscope.

<(1) Preparation of Mouse Macrophage Culture Supernatant Sample>

In order to obtain peritoneal macrophage, 2 mL of a 3% thioglycolatesolution (produced by Fluka) was injected intraperitoneally to 6 femaleC57BL/6J mice, 8 weeks old (purchased from Japan SLC, Inc.). After 3days, macrophages were recovered from the peritoneal cavity, and therecovered macrophages were cultured for 2 days in 4 pieces of 150 mmdishes for cell culture, using 80 mL of DMEM (produced by NacalaiTesque, Inc.) containing 10% FBS (produced by BioWest Co., Ltd.), thenthe culture supernatant was recovered to obtain the mouse macrophageculture supernatant sample.

<(2) Preparation of Tim-4 Carrier>

The mTim-4 carrier, where the SH-group biotin-labeled Fc tag fusion typemTim-4 protein is bound on the Dynabeads MyOne Streptavidin C1, wasprepared by the same method as in Examples 11 to 12, except for using 1mg of the Dynabeads MyOne Streptavidin C1 (produced by Thermo FisherScientific Inc.), instead of 0.6 mg of the Dynabeads MyOne StreptavidinC1 (produced by Thermo Fisher Scientific Inc.), using 100 μL of the PBSsolution containing 100 μg of the SH-group biotin labeled Fc tag fusiontype mTim-4 protein, instead of 200 μL of the PBS solution containing 1μg of the SH-group biotin labeled Fc tag fusion type mTim-4 protein, andsetting reaction time of the PBS solution containing the Dynabeads andthe SH-group biotin labeled Fc tag fusion type mTim-4 protein to 2 hoursinstead of 1 hour.

<(3) Obtaining of Extracellular Membrane Vesicles Using Tim-4 Carrier>

The mouse macrophage culture supernatant sample (32 mL) was subjected tocentrifugal separation treatment (first time: 800×G, for 10 minutes,second time: 12,000×G, for 30 minutes) to obtain the supernatant. AfterCaCl₂ was added to the resulting supernatant, so as to attain the finalconcentration of 2 mM, 1 mg of the mTim-4 carrier prepared above wasadded, and mixed while stirring at room temperature for 1 hour.Subsequently, the mTim-4 carrier, mixed under stirring at roomtemperature for 1 hour, was recovered, then subjected to the washingoperation three times with 5 mL of TBS-T containing 2 mM CaCl₂ as thefinal concentration, and further subjected to the washing operationtwice with 1 mL thereof, and then eluted three times with 100 μL of TBScontaining 1 mM EDTA to obtain 300 μL of the extracellular membranevesicles fraction.

Subsequently, in order to surely recover the extracellular membranevesicles in the mouse macrophage culture supernatant sample, theextracellular membrane vesicles were recovered again by the followingmethod, from the mouse macrophage culture supernatant sample aftereluting three times with 100 μL of TBS containing 1 mM EDTA to obtain300 μL of the extracellular membrane vesicles fraction. That is, CaCl₂was added to the mouse macrophage culture supernatant sample, afterthree times of the elutions with 100 μL of TBS containing 1 mM EDTA, soas to attain the final concentration of 2 mM, then 1 mg of the mTim-4carrier, after the elution of three times with 100 μL of TBS containing1 mM EDTA, was added and mixed while stirring at room temperature for 1hour. Subsequently, the mTim-4 carrier, mixed under stirring at roomtemperature for 1 hour, was recovered, and subjected to the washingoperation three times with 5 mL of TBS-T containing 2 mM CaCl₂ at thefinal concentration, and further subjected to the washing operationtwice with 1 mL thereof, and then eluted three times with 100 μL of TBScontaining 1 mM EDTA to obtain 300 μL of the extracellular membranevesicles fraction.

Thereafter, in order to more surely recover the extracellular membranevesicles in the mouse macrophage culture supernatant sample, the aboveoperation was carried out again, and the extracellular membrane vesicleswere recovered from the mouse macrophage culture supernatant sample,after the elution of six times in total with 100 μL of TBS containing 1mM EDTA to obtain 300 μL of the extracellular membrane vesiclesfraction.

Total 900 μL of the extracellular membrane vesicles, obtained by ninetimes of the elution operations, was concentrated to 60 μL using anAmicon Ultra-0.5 mL 10K centrifugal filter column to obtain a sample forobservation with an electron microscope.

<(4) Observation of Extracellular Membrane Vesicles Obtained byObtaining Method of the Present Invention, with Electron Microscope>

The sample (10 μL) for observation with the electron microscope wasadsorbed on a grid, and the excessive sample for observation with theelectron microscope was suctioned with a filter paper. Subsequently, thegrid was washed with water, and stained twice with a uranium acetateaqueous solution, and then observation by a negative staining method wascarried out with a transmission electron microscope.

It should be noted that the type of the mTim-4 protein, the carrier, andthe eluent used for obtaining the extracellular membrane vesicles fromthe mTim-4 carrier, used in Example 21, are shown in the following Table9.

TABLE 9 Example 21 Mouse-derived Type of tag Fc tag Tim-4 carrier Tagfusion type Biotin-labeled mouse-derived (SH-group) Tim-4 proteinCarrier Streptavidin beads Eluent TBS containing 1 mM EDTA<Results>

The resulting observation image with the electron microscope is shown inFIG. 5. From FIG. 5, it has been revealed that the extracellularmembrane vesicles, having a diameter of about 50 to 150 nm, can beobtained, while maintaining a spherical shape, according to theobtaining method of the present invention.

From the above, it has been revealed that the extracellular membranevesicles can be obtained in an intact state, according to the obtainingmethod of the present invention.

Examples 22 to 25. Obtaining of Extracellular Membrane Vesicles by Tim-4Carrier of the Present Invention (Obtaining Method of the PresentInvention)

The extracellular membrane vesicles pertaining to the present inventionwas obtained using the carrier where the human-derived Tim-4 protein wasimmobilized on the beads (hereinafter it may be abbreviated as “thehTim-4 carrier”, in some cases), and the mTim-4 carrier.

<(1) Preparation of Culture Supernatant Sample>

Preparation of the culture supernatant sample was carried out in thesame method as in “(1) preparation of culture supernatant sample” ofExamples 1 to 8.

<(2) Dilution of Fc Tag Fusion Type Tim-4 Protein>

The PBS solution containing 100 μg/mL of the biotin-unlabeled Fc tagfusion type human-derivedTim-4 protein was prepared by dissolving, in 1mL of PBS, 100 μg of a freeze-dried product of the biotin-unlabeled Fctag fusion type human-derived Tim-4 protein (produced by Wako PureChemical Industries, Ltd., SEQ ID NO: 7 (the human-derived Tim-4 proteinin which Fc tag was fused to the N-terminal 25 to 315 amino acid regionof the human-derived Tim-4 protein (GenBank NP_612388.2)). The PBSsolution (200 μL) containing 1 μg of the biotin-unlabeled Fc tag fusiontype hTim-4 protein was obtained by mixing 10 μL of the PBS solutioncontaining the biotin-unlabeled Fc tag fusion type hTim-4 protein, and190 μL of PBS.

In addition, the PBS solution containing 100 μg/mL of thebiotin-unlabeled Fc tag fusion type mTim-4 protein was prepared bydissolving, in 1 mL of PBS, 100 μg of the freeze-dried product of thebiotin-unlabeled Fc tag fusion type mouse-derived Tim-4 protein(produced by Wako Pure Chemical Industries, Ltd., SEQ ID NO: 6 (themTim-4 protein in which the Fc tag was fused to N-terminal 22 to 279amino acid region of the mouse-derived Tim-4 protein (GenBankNP_848874.3)). The PBS solution (200 μL) containing 1 μg of thebiotin-unlabeled Fc tag fusion type mTim-4 protein was obtained bymixing 10 μL of the PBS solution containing the biotin-unlabeled Fc tagfusion type mTim-4 protein, and 190 μL of PBS.

<(3) Washing of Beads>

The PBS-T solution (20 μL) containing the 30 μg/μL Dynabeads Protein G(produced by Thermo Fisher Scientific Inc.) (containing 0.6 mg of theDynabeads Protein G) was dispensed into each of two 1.5 mL tubes(manufactured by BM Equipment Co., Ltd.) to carry out the washingoperation using 500 μL of PBS.

<(4) Immobilization of Biotin-Unlabeled Fc Tag Fusion Type Tim-4 Proteinon Beads>

For one of two 1.5 mL tubes containing the Dynabeads Protein G (0.6 mg),200 μL of the PBS solution containing 1 μg of the biotin-unlabeled Fctag fusion type hTim-4 protein, prepared above, was added in itsentirety, and subjected to a reaction at 8° C. for 1 hour to obtain 200μL of the PBS solution containing the carrier on which thebiotin-unlabeled Fc tag fusion type hTim-4 protein was bound (the hTim-4carrier).

For remaining one 1.5 mL tube containing the Dynabeads Protein G (0.6mg), 200 μL of the PBS solution containing 1 μg of the biotin-unlabeledFc tag fusion type mTim-4 protein prepared above was added in itsentirety, and subjected to a reaction at 8° C. for 1 hour to obtain 200μL of the PBS solution containing the carrier on which thebiotin-unlabeled Fc tag fusion type mTim-4 protein was bound (mTim-4carrier).

<(5) Obtaining of Extracellular Membrane Vesicles by Obtaining Method ofthe Present Invention>

After subjecting each 0.6 mg of the resulting hTim-4 carrier and themTim-4 carrier above to the washing operation with 500 μL of PBS threetimes, 200 μL of the calcium ion-containing culture supernatant samplewas added to each of the hTim-4 carrier and the mTim-4 carrier, in apellet state, and subjected to a reaction each at 8° C. for 3 hours.Thereafter, the hTim-4 carrier and the mTim-4 carrier after the reactionwere each subjected to the washing operation three times with 500 μL of2 mM CaCl₂-containing TBS-T.

At the third time of washing, the hTim-4 carrier and the mTim-4 carriereach 250 μL, (0.3 mg of the carrier) were dispensed into two 1.5 mLtubes. To each 0.3 mg of the mTim-4 carrier and the hTim-4 carrier, in apellet state, 20 μL of the 1% SDS aqueous solution, or the 1 mM EDTAaqueous solution was added, as the eluent, and then they were mixed atroom temperature for 10 seconds using a vortex mixer and spun down. The1.5 mL tubes were each loaded on a magnet stand, and the hTim-4 carrierand the mTim-4 carrier were collected on the tube wall by magnetic forceto recover each supernatant (eluate).

It should be noted that the types of the Tim-4 proteins, the carrierused, and the eluates used for obtaining the extracellular membranevesicles from the Tim-4 carrier, in Examples 22 to 25, and the lanenumbers in Western blotting to be described later are shown in thefollowing Table 10.

TABLE 10 Example 22 Example 23 Example 24 Example 25 Mouse-derived Tim-4protein Fc tag fusion type Fc tag fusion type Tim-4 carrier orhuman-derived mouse-derived Human-derived Tim-4 protein Tim-4 proteinTim-4 carrier (Biotin-unlabeled) (Biotin-unlabeled) Carrier DynabeadsProtein G beads Eluent 1% 1 mM 1% 1 mM SDS EDTA SDS EDTA Lane number inFIG. 6 1 2 3 4<(6) Western Blotting>

Western blotting was carried out by the same method as in “(7) Westernblotting” of Examples 1 to 8, except for using “7.5 μL of eachsupernatant (eluate) obtained in Examples 22 to 25 (above (5)”, insteadof “7.5 μL of each supernatant (eluate) obtained in Examples 1 to 8”.

<Results>

The results of Western blotting obtained are shown in FIG. 6. In FIG. 6,each lane is as follows:

Lane 1: Results of Example 22 (the results of the case of using thehTim-4 carrier where the biotin-unlabeled Fc tag fusion type hTim-4protein was bound on the Protein G beads, and using the 1% SDS aqueoussolution as the eluent);

Lane 2: Results of Example 23 (the results of the case of using thehTim-4 carrier where the biotin-unlabeled Fc tag fusion type hTim-4protein was bound on the Protein G beads, and using the 1 mM EDTAaqueous solution as the eluent);

Lane 3: Results of Example 24 (the results of the case of using themTim-4 carrier where the biotin-unlabeled Fc tag fusion type mTim-4protein was bound on the Protein G beads, and using the 1% SDS aqueoussolution as the eluent);

Lane 4: Results of Example 25 (the results of the case of using themTim-4 carrier where the biotin-unlabeled Fc tag fusion type mTim-4protein was bound on the Protein G beads, and using the 1 mM EDTAaqueous solution as the eluent).

From FIG. 6, it has been revealed that the extracellular membranevesicles including exosome can be obtained, according to the obtainingmethod of the present invention, from the fact that a band of Lamp-1,which is an exosome marker, was observed at the vicinity of 100 kDa inany case of Examples 22 to 25 (Example 22 to 25: the lanes 1 to 4).

In addition, it has been revealed that the Tim-4 protein can be used forthe Tim-4 carrier of the present invention and the obtaining method ofthe present invention, regardless of organism species of origin.

Examples 26 to 27 and Comparative Examples 4 to 9. Comparison ofObtained Amount of Extracellular Membrane Vesicles by Tim-4 Carrier andCarrier on which PS Protein was Immobilized

Obtaining of the extracellular membrane vesicles pertaining to thepresent invention was carried out using the Tim-4 carrier and thecarrier where PS proteins (human origin Annexin V, human MFG-E8, andmouse derived MFG-E8) were each immobilized on the beads.

<(1) Dilution of PS Protein-Containing PBS Solution>

The His tag fusion type human-derived Annexin V (20 μg, produced byCreative BioMart Co., Ltd., it may be abbreviated as “the His tag fusiontype hAnnexin V”, in some cases) was dissolved in 100 μL of PBS, so asto attain 200 μg/mL to prepare the His tag fusion type hAnnexinV-containing PBS solution. Then, 5 μL of the PBS solution containing 1μg of the His tag fusion type hAnnexin V protein and 195 μL of PBS weremixed for dilution.

The His tag fusion type human-derived MFG-E8 (50 μg, produced by R & DSystems Co., Ltd., it may be abbreviated as “the His tag fusion typehMFG-E8”) was dissolved in 500 μL of PBS, so as to attain 100 μg/mL toprepare the His tag fusion type hMFG-E8-containing PBS solution. Then,10 μL of the PBS solution containing 1 μg of the His tag fusion typehMFG-E8 protein, and 190 μL of PBS were mixed for dilution. The His tagfusion type mouse-derived MFG-E8 (50 μg, produced by R & D Systems Co.,Ltd., it may be abbreviated as “the His tag fusion type mMFG-E8”) wasdissolved in 500 μL of PBS, so as to attain 100 μg/mL to prepare the Histag fusion type mMFG-E8-containing PBS solution. Then, 10 μL of the PBSsolution containing 1 μg of the His tag fusion type mouse-derived MFG-E8protein, and 190 μL of PBS were mixed for dilution.

<(2) Preparation of Anti-his Tag Antibody-Immobilized Beads>

A solution (100 μL) containing 30 μg/μL Dynabeads M-270 Carboxylic Acid(produced by Thermo Fisher Scientific Inc.) (containing 3 mg of theDynabeads M-270 Carboxylic Acid) was dispensed into the 1.5 mL tube(manufactured by BM Equipment Co., Ltd.), and the washing operation wascarried out using a reaction buffer (0.1 M MES, pH 5.0).

Subsequently, 60 μL of a solution of a 6× His antibody (produced by WakoPure Chemical Industries, Ltd.) (containing 60 μg of the 6× Hisantibody), diluted with 490 μL of the reaction buffer, was added andmixed with inversion at room temperature for 30 minutes, then 50 μL of 6mg/mL WSC (produced by Dojindo Molecular Technologies, Inc.) was addedand mixed with inversion at room temperature for 4 hours. The DynabeadsM-270 Carboxylic Acid after mixing by inversion was subjected to thewashing operation with TBS-T, and diluted with 100 μL of PBS to obtain 3mg of the anti-His tag antibody-immobilized beads.

<(3) Preparation of Tim-4 Carrier and Carrier on which Various PSProteins were Immobilized>

Subsequently, 100 μL of the PBS solution containing the resultinganti-His tag antibody-immobilized beads (containing 3 mg of the anti-Histag antibody-immobilized beads) was dispensed into each of four 1.5 mLtubes (manufactured by BM Equipment Co., Ltd.) in an amount of each 20μL (containing 0.6 mg of the anti-His tag antibody-immobilized beads),and the washing operation was each carried out with 500 μL PBS.

After that, for one of the four 1.5 mL tubes containing 0.6 mg of theanti-His tag antibody-immobilized beads in a pellet state, after thewashing operation, 200 μL of the His tag fusion type mTim-4protein-containing PBS solution prepared above (containing 1 μg of theHis tag fusion type mTim-4 protein) was added and subjected to areaction at 8° C. for 1 hour.

Further, for one tube, 200 μL of the His tag fusion type hAnnexinV-containing PBS solution prepared above (containing 1 μg of the His tagfusion type hAnnexin V) was added and subjected to a reaction at 8° C.for 1 hour.

For one tube, 200 μL of the His tag fusion type hMFG-E8-containing PBSsolution prepared above (containing 1 μg of the His tag fusion typehMFG-E8) was added and subjected to a reaction at 8° C. for 1 hour.

For one tube, 200 μL of the His tag fusion type mMFG-E8-containing PBSsolution prepared above (containing 1 μg of the His tag fusion typemMFG-E8) was added and subjected to a reaction at 8° C. for 1 hour.

In this way, 200 μL of the PBS solutions each containing 0.6 mg of eachof the four types of the carriers shown in the following Table 11 wereobtained.

TABLE 11 1 2 3 4 Mouse-derived Tim-4 Phosphatidyl- His tag fusion typeHis tag fusion type His tag fusion type His tag fusion type carrierserine bound mouse-derived human-derived human-derived mouse-derived orprotein Tim-4 protein Annexin V MFG-E8 MFG-E8 Phosphatidylserine CarrierAnti-Histag antibody-immobilized beads bound protein- immobilizedcarrier<(4) Obtaining of Extracellular Membrane Vesicles>

The PBS solution (200 μL) each containing 0.6 mg of each of theresulting 4 types of carriers above was subjected to the washingoperation three times with 500 μL of PBS, then 200 μL of the calciumion-containing culture supernatant sample, prepared by the same methodas in Examples 1 to 8, was added to each of 5 types of the carriers in apellet state, and subjected to a reaction at 8° C. for 3 hours.

Thereafter, 4 types of the carriers, which were subjected to a reactionwith the calcium ion-containing culture supernatant sample, weresubjected to the washing operation three times each with 500 μL of TBS-Tcontaining 2 mM CaCl₂.

At the third time of washing, each 250 μL of 4 types of the carriers wasdispensed into two 1.5 mL tubes. The 1% SDS aqueous solution or the 1 mMEDTA aqueous solution (each 20 μL) was added, as the eluent, to each 0.3mg of 4 types of the carriers in a pellet state, and then spun downusing a vortex mixer at room temperature for 10 seconds. Each 1.5 mL ofthe tube was loaded on a magnet stand, and the resulting 4 types of thecarriers were collected on the tube wall using magnetic force to recoverthe supernatant (eluate).

It should be noted that types of the PS proteins, the carriers, and theeluates used for obtaining the extracellular membrane vesicles from thecarriers, used in each Example, and the lane numbers in Western blottingto be described later are shown in the following Table 12.

TABLE 12 Comparative Comparative Comparative Comparative ComparativeComparative Example 26 Example 27 Example 4 Example 5 Example 6 Example7 Example 8 Example 9 Mouse-derived PS protein His tag fusion type Histag fusion type His tag fusion type His tag fusion type Tim-4 carriermouse-derived Tim-4 human-derived Annexin human-derived mouse-derived orprotein V MFG-E8 MFG-E8 PS protein- Carrier Anti-His tagantibody-immobilized beads immobilized carrier Eluent 1% 1 mM 1% 1 mM 1%1 mM 1% 1 mM SDS EDTA SDS EDTA SDS EDTA SDS EDTA Lane number in FIG. 7 12 3 4 5 6 7 8<(5) Western Blotting>

Western blotting was carried out by the same method as in “(7) Westernblotting” of Examples 1 to 8, except for using “7.5 μL of eachsupernatant (eluate) obtained in Examples 26 to 27, Comparative Examples4 to 9 (above (4))”, instead of “7.5 μL of each supernatant (eluate)obtained in Examples 1 to 8”.

<Results>

The results of Western blotting obtained are shown in FIG. 7. In FIG. 7,each lane is as follows:

Lane 1: Results of Example 26 (the results of the case of using thecarrier where the His tag fusion type mTim-4 protein was bound on theanti-His tag antibody-immobilized beads, and using the 1% SDS aqueoussolution as the eluent);

Lane 2: Results of Example 27 (the results of the case of using thecarrier where the His tag fusion type mTim-4 protein was bound on theanti-His tag antibody-immobilized beads, and using the 1 mM EDTA aqueoussolution as the eluent);

Lane 3: Results of Comparative Example 4 (the results of the case ofusing the carrier where the His tag fusion type hAnnexin V was bound onthe anti-His tag antibody-immobilized beads, and using the 1% SDSaqueous solution as the eluent);

Lane 4: Results of Comparative Example 5 (Results in a case of using thecarrier where the His tag fusion type hAnnexin V was bound on theanti-His tag antibody-immobilized beads, and using the 1 mM EDTA aqueoussolution as the eluent);

Lane 5: Results of Comparative Example 6 (the results of the case ofusing the carrier where the His tag fusion type hMFG-E8 was bound on theanti-His tag antibody-immobilized beads, and using the 1% SDS aqueoussolution as the eluent);

Lane 6: Results of Comparative Example 7 (the results of the case ofusing the carrier where the His tag fusion type hMFG-E8 was bound on theanti-His tag antibody-immobilized beads, and using the 1 mM EDTA aqueoussolution as the eluent);

Lane 7: Results of Comparative Example 8 (the results of the case ofusing the carrier where the His tag fusion type mMFG-E8 was bound on theanti-His tag antibody-immobilized beads and using the 1% SDS aqueoussolution as the eluent);

Lane 8: Results of Comparative Example 9 (the results of the case ofusing the carrier where the His tag fusion type mMFG-E8 was bound on theanti-His tag antibody-immobilized beads, and using the 1 mM EDTA aqueoussolution as the eluent).

From FIG. 7, it has been revealed that the extracellular membranevesicles including exosome can be obtained, according to the obtainingmethod of the present invention, from the fact that a band of Lamp-1,which is an exosome marker, was observed at the vicinity of 100 kDa inany case of Examples 26 to 27 (Example 26 to 27: the lanes 1 to 2).

On the other hand, from FIG. 7, it has been revealed that theextracellular membrane vesicles including exosome cannot be obtained bythe carriers on which the PS proteins other than the Tim-4 protein(Annexin V, MFG-E8) were immobilized, from the fact that the band ofLamp-1 was not observed at the vicinity of 100 kDa in any case ofComparative Examples 4 to 9 (Comparative Example 4 to 9: the lanes 3 to8).

Examples 28 to 33. Obtaining of Extracellular Membrane Vesicles byObtaining Method of the Present Invention (Examination of ComplexFormation Step)

The extracellular membrane vesicles were obtained by each of theobtaining methods of the present invention as described below.

In Examples 28 to 29, after the SH-group biotin-labeled FLAG tag fusiontype mouse-derived Tim-4 protein, prepared by the same method as inExamples 13 to 14, was subjected to a reaction with the carrierpertaining to the present invention, the sample was further addedthereto to obtain the extracellular membrane vesicles pertaining to thepresent invention.

In Examples 30 to 31, after the SH-group biotin-labeled FLAG tag fusiontype mouse-derived Tim-4 protein, prepared by the same method as inExamples 13 to 14, was subjected to a reaction with the sample, thecarrier pertaining to the present invention was further added thereto toobtain the extracellular membrane vesicles pertaining to the presentinvention.

In Examples 32 to 33, the SH-group biotin-labeled FLAG tag fusion typemouse-derived Tim-4 protein, prepared by the same method as in Examples13 to 14, the sample, and the carrier pertaining to the presentinvention were added at the same time to obtain the extracellularmembrane vesicles pertaining to the present invention.

<Washing of Beads>

Each 60 μL of the PBS solution containing the 10 μg/μL Dynabeads M-270Streptavidin C1 (produced by Thermo Fisher Scientific Inc.) (containing0.6 mg of the Dynabeads M-270 Streptavidin C1) was dispensed into three1.5 mL tubes (manufactured by BM Equipment Co., Ltd.), and the washingoperation was carried out each using 500 μL of PBS.

The His tag fusion type mTim-4 protein-containing PBS solution (270 μL)(containing 50 μg of the His tag fusion type mTim-4 protein) was labeledwith biotin by the same method as in Examples 13 to 14 to obtain 200 μLof the PBS solution containing 39.2 μg of the SH-group biotin-labeledFLAG tag fusion type mTim-4 protein. Into 5.1 μL of the PBS solutioncontaining 1 μg of the resulting SH-group biotin-labeled FLAG tag fusiontype mTim-4 protein, 194.9 μL of the PBS solution was added to obtain200 μL of the PBS solution containing 1 μg of the SH-groupbiotin-labeled FLAG tag fusion type mTim-4 protein.

<The Case of Subjecting the SH-Group Biotin-Labeled FLAG Tag Fusion TypemTim-4 Protein and the Carrier Pertaining to the Present Invention to aReaction, Followed by Further Subjecting the Extracellular MembraneVesicles in the Sample Pertaining to the Present Invention to a Reaction(Examples 28 to 29)>

The PBS solution (200 μL) containing 1 μg of the SH-group biotin-labeledFLAG tag fusion type mTim-4 protein was added to the 1.5 mL tubecontaining 0.6 mg of the pellet-like Dynabeads M-270 Streptavidin C1which had been subjected to the washing operation, and the DynabeadsM-270 Streptavidin C1, and the SH-group biotin-labeled FLAG tag fusiontype mTim-4 protein were brought into contacted and subjected to areaction at 8° C. for 1 hour to obtain the mTim-4 carrier. Subsequently,the resulting mTim-4 carrier was subjected to the washing operationthree times with 500 μL of PBS, and 200 μL of the calcium ion-containingculture supernatant sample, prepared by the same method as in Example 1,was added to the 1.5 mL tube containing 0.6 mg of the mTim-4 carrierafter washing, and subjected to a reaction at 8° C. for 3 hours. Afterthat, the mTim-4 carrier which had been subjected to a reaction with thecalcium ion-containing culture supernatant sample was subjected to thewashing operation three times with 500 μL of TBS-T added with 2 mM CaCl₂as the final concentration. At the third time of washing, each 250 μL ofthe mTim-4 carrier was dispensed into two 1.5 mL tubes. After theaddition of 20 μL of the 1% SDS aqueous solution or the 1 mM EDTAaqueous solution, as the eluent, to each 0.3 mg of the mTim-4 carrier ina pellet state, they were mixed at room temperature for 10 seconds usinga vortex mixer and spun down. Each of the 1.5 mL tubes was loaded on amagnet stand, and the mTim-4 carrier was collected on the tube wallusing magnetic force to recover each supernatant (eluate).

<The Case of Subjecting SH-Group Biotin-Labeled FLAG Tag Fusion TypemTim-4 Protein and Sample Pertaining to the Present Invention to aReaction, Followed by Further Subjecting the Carrier Pertaining to thePresent Invention to a Reaction Examples 30-31>

The calcium ion-containing culture supernatant sample (200 μL) preparedby the same method as in Example 1 was dispensed into the 1.5 mL tube,and 5.1 μL of the PBS solution containing 1 μg of the SH-groupbiotin-labeled FLAG tag fusion type mTim-4 protein was added, and theextracellular membrane vesicles in the calcium ion-containing culturesupernatant sample, and the SH-group biotin-labeled FLAG tag fusion typemTim-4 protein were brought into contacted and subjected to a reactionat 8° C. for 3 hours.

The complex of the extracellular membrane vesicles and the mTim-4carrier was obtained by the addition of the solution after subjectingthe extracellular membrane vesicles in the calcium ion-containingculture supernatant sample, and the SH-group biotin-labeled FLAG tagfusion type mTim-4 protein to a reaction at 8° C. for 3 hours, to the1.5 mL tube containing 0.6 mg of the pellet-like Dynabeads M-270Streptavidin C1 after the washing operation, and subjecting them to areaction at 8° C. for 1 hour. Thereafter, the resulting complex of theextracellular membrane vesicles and the mTim-4 carrier was subjected tothe washing operation three times with 500 μL of TBS-T added with 2 mMCaCl₂ as the final concentration. At the third time of washing, each 250μL of the complex with the mTim-4 carrier was dispensed into the two 1.5mL tubes. After the addition of 20 μL of the 1% SDS aqueous solution orthe 1 mM EDTA aqueous solution, as the eluent, to each 0.3 mg of thecomplex with the mTim-4 carrier in a pellet state, they were mixed usinga vortex mixer at room temperature for 10 seconds and spun down. Each ofthe 1.5 mL tubes was loaded on a magnet stand, and the mTim-4 carrierwas collected on the tube wall using magnetic force to recover eachsupernatant (eluate).

<The Case of Subjecting the SH-Group Biotin-Labeled FLAG Tag Fusion TypemTim-4 Protein, Sample Pertaining to the Present Invention, and CarrierPertaining to the Present Invention to Reaction Simultaneously (Examples32 to 33)>

The calcium ion-containing culture supernatant sample (200 μL) preparedby the same method as in Example 1, and 5.1 μL of the PBS solutioncontaining 1 μg of the SH-group biotin-labeled FLAG tag fusion typemTim-4 protein were added to the 1.5 mL tube containing 0.6 mg of thepellet-like Dynabeads M-270 Streptavidin C1 after the washing operation,and subjected to a reaction at 8° C. for 4 hours. The Dynabeads M-270Streptavidin C1 in a pellet state after the reaction was subjected tothe washing operation three times with 500 μL of TBS-T containing 2 mMCaCl₂ as the final concentration. At the third time of washing, each 250μL of the mTim-4 carrier was dispensed into the two 1.5 mL tubes, and 20μL of the 1% SDS aqueous solution or the 1 mM EDTA aqueous solution wasadded, as the eluate, to each 0.3 mg of the mTim-4 carrier, and thenthey were mixed using a vortex mixer at room temperature for 10 secondsand spun down. Each of the 1.5 mL tubes was loaded on a magnet stand,and the Dynabeads M-270 Streptavidin C1 was collected on the tube wallusing magnetic force to recover each supernatant (eluate).

It should be noted that, the order of bringing into contacted thecarrier, the mTim-4 protein, and the calcium ion-containing culturesupernatant sample, and the types of the eluents used for obtaining theextracellular membrane vesicles from the carrier, in each Example, areshown in the following Table 13.

TABLE 13 Order of contact Eluent Example 28 (1) Contacting carrier withmouse-derived 1% SDS Tim-4 protein Example 29 (2) Further contactingwith calcium ion- 1% EDTA containing cell culture supernatant sampleExample 30 (1) Contacting mouse-derived Tim-4 protein 1% SDS withcalcium ion-containing cell culture Example 31 supernatant sample 1%EDTA (2) Further contacting with carrier Example 32 (1) Contactingcarrier, calcium ion-containing 1% SDS cell culture supernatant sample,and Example 33 mouse-derived Tim-4 protein at the same time 1% EDTA<Western Blotting>

Western blotting was carried out by the same method as in “(7) Westernblotting” of Examples 1 to 8, except for using “7.5 μL of eachsupernatant (eluate) obtained in Examples 28 to 33 (above (5))”, insteadof “7.5 μL of each supernatant (eluate) obtained in Examples 1 to 8”.

<Results>

The results of Western blotting obtained are shown each in FIG. 8. InFIG. 8, each lane is as follows:

Lane 1: Results of Example 28 (the results of the case of obtaining theextracellular membrane vesicles by the method for bringing intocontacted the carrier with the mTim-4 protein, further bringing intocontacted with the calcium ion-containing cell culture supernatantsample, and using the 1% SDS aqueous solution as the eluent);

Lane 2: Results of Example 29 (the results of the case of obtaining theextracellular membrane vesicles by the method for bringing intocontacted the carrier with the mTim-4 protein, further bringing intocontacted with the calcium ion-containing cell culture supernatantsample, and using the 1 mM EDTA aqueous solution was used as an eluent);

Lane 3: Results of Example 30 (the results of the case of obtaining theextracellular membrane vesicles by the method for bringing intocontacted the mTim-4 protein and the calcium ion-containing cell culturesupernatant sample, further bringing into contacted with the carrier,and using the 1% SDS aqueous solution was used as an eluent);

Lane 4: Results of Example 31 (the results of the case of obtaining theextracellular membrane vesicles by the method for bringing into themTim-4 protein with the calcium ion-containing cell culture supernatantsample, further bringing into contacted with the carrier, and using the1 mM EDTA aqueous solution was used as an eluent);

Lane 5: Results of Example 32 (the results of the case of obtaining theextracellular membrane vesicles by the method for bringing intocontacted the carrier, the calcium ion-containing cell culturesupernatant sample, and the mTim-4 protein at the same time, and usingthe 1% SDS aqueous solution as the eluent),

Lane 6: Results of Example 33 (the results of the case of obtaining theextracellular membrane vesicles by the method for bringing intocontacted the carrier, the calcium ion-containing cell culturesupernatant sample, and the mTim-4 protein at the same time, and usingthe 1 mM EDTA aqueous solution as the eluent).

As shown in FIG. 8, a band of Lamp-1, which is an exosome marker, wasobtained at the vicinity of 100 kDa in any case of Examples 28 to 33(Examples 28 to 33: the lanes 1 to 6).

In addition, from the results of Examples 28 to 33, it has been revealedthat the step for forming the complex of the Tim-4 protein bound to thecarrier and the extracellular membrane vesicles in the sample (complexformation step) may be any step, as long as the complex of the mTim-4protein bound to the carrier and the extracellular membrane vesicles inthe sample is accordingly formed, regardless of the order of contactamong the Tim-4 protein pertaining to the present invention, the carrierpertaining to the present invention, and the extracellular membranevesicles in the sample.

Examples 34 to 35, Comparative Examples 10 to 13. Comparison BetweenObtaining Methods of the Present Invention and Conventional Method

As described below, the extracellular membrane vesicles were obtained bythe obtaining method of the present invention by elution with SDS(Example 34), by the obtaining method of the present invention byelution with EDTA (Example 35), by the anti-CD 63 antibodyimmobilization method (Comparative Examples 10 to 11), and byExosome-Human CD63 Isolation/Detection (Comparative Examples 12 to 13).

<Immobilization of Tim-4 Protein on Carrier>

The solution (15 μL) containing the 10 μg/μL Dynabeads M-270Streptavidin (produced by Thermo Fisher Scientific Inc.) (containing1×10⁷ particles of the Dynabeads M-270 Streptavidin) was dispensed intothe 1.5 mL tube (manufactured by BM Equipment Co., Ltd), and subjectedto the washing operation.

Subsequently, 200 μL of the PBS solution containing 0.25 μg of theSH-group biotin-labeled FLAG tag fusion type mouse-derived Tim-4protein, prepared by the same method as in Examples 11 to 12, was addedin its entirety to the 1.5 mL tube containing the Dynabeads M-270Streptavidin after the washing operation, and subjected to a reactionunder refrigeration for 1 hour to obtain the PBS solution containing themTim-4 carrier.

<Preparation of Anti-CD63 Antibody (H5C6)-Immobilized Carrier>

The solution (50 μL) containing the 30 μg/μL Dynabeads M-270 CarboxylicAcid (produced by Thermo Fisher Scientific, Inc.) (containing 1×10⁸particles of the Dynabeads M-270 Carboxylic Acid) was dispensed into the1.5 mL tube (manufactured by BM Equipment Co., Ltd), and subjected tothe washing operation using the reaction buffer (0.1 M MES, pH 5.0).

On the other hand, 30 g (60 μL) of the anti-CD63 antibody (H5C6)(produced by BD Pharmingen) was diluted with 490 μL of the reactionbuffer (0.1 M MES, pH 5.0) to obtain a reaction buffer-diluted anti-CD63 antibody solution.

Subsequently, 550 μL of the reaction buffer-diluted anti-CD 63 antibodysolution was added to the 1.5 mL tube containing the Dynabeads M-270Carboxylic Acid, after the washing operation, and mixed with inversionat room temperature for 30 minutes. Thereafter, further 50 μL of 3 mg/mLWSC (produced by Dojindo Molecular Technologies, Inc.) was added andmixed with inversion at room temperature for 4 hours, and after thewashing operation with TBS-T, dilution was carried out with 50 μL PBS toobtain the PBS solution containing the anti-CD63 antibody (H5C6)carrier.

In this way, two kinds of the carriers shown in the following Table 14were obtained.

TABLE 14 1 2 Protein or antibody Biotin-labeled (SH-group) Anti-CD63 tobe immobilized FLAG tag fusion type antibody (H5C6) on carriermouse-derived Tim-4 protein Carrier Dynabeads M-270 Dynabeads M-270Streptavidin Carboxylic Acid<Obtaining of Extracellular Membrane Vesicles by Obtaining Method of thePresent Invention>

The resulting mTim-4 carrier-containing PBS solution (200 μL) above(containing 1×10⁷ particles of the mTim-4 carriers) was subjected to thewashing operation three times with 500 μL of PBS, and then 50 μL of thecalcium ion-containing culture supernatant sample, prepared by the samemethod as in Examples 1 to 8, was added to the mTim-4 carrier in apellet state, and subjected to a reaction at 8° C. for 3 hours.

Thereafter, the mTim-4 carrier after the reaction was subjected to thewashing operation three times with 500 μL of TBS-T containing 2 mMCaCl₂. At the third time of washing, each 250 μL of the mTim-4 carrierwas dispensed into the two 1.5 mL tubes. Each 20 μL of the 1% SDSaqueous solution or the 1 mM EDTA aqueous solution was added, as theeluent, to each 0.3 mg of the mTim-4 carrier in a pellet state, and theywere mixed using a vortex mixer at room temperature for each 10 secondsand spun down. Each of the 1.5 mL tubes was loaded on a magnet stand,and the mTim-4 carrier was collected on the tube wall using magneticforce to recover the supernatant (eluate).

<Obtaining of Extracellular Membrane Vesicles by Anti-CD 63 AntibodyImmobilization Method>

The extracellular membrane vesicles in the calcium ion-containingculture supernatant sample was obtained in the same method as in the<Obtaining of extracellular membrane vesicles by obtaining method of thepresent invention>, except for using “5 μL of the anti-CD63 antibody(H5C6) carrier-containing PBS solution (containing 1×10⁷ particles ofthe carrier)”, instead of “200 μL of the mTim-4 carrier-containing PBSsolution”, and by eluting the extracellular membrane vesicles with the1% SDS aqueous solution or the 1 mM EDTA aqueous solution to recover thesupernatant (eluate).

<Obtaining of Extracellular Membrane Vesicles by Exosome-Human CD 63Isolation/Detection>

The extracellular membrane vesicles in the calcium ion-containingculture supernatant sample was obtained by the same method as in the<Obtaining of extracellular membrane vesicles by obtaining method of thepresent invention>, except for using “1 mL of the Exosome-Human CD 63Isolation/Detection (produced by Thermo Fisher Scientific Inc.)(containing 1×10⁷ particles of the carrier)”, instead of “200 μL of themTim-4 carrier-containing PBS solution”, and by eluting theextracellular membrane vesicles with the 1% SDS aqueous solution or the1 mM EDTA aqueous solution to recover the supernatant (eluate).

It should be noted that, the types of the carriers, and the eluents usedto obtain the extracellular membrane vesicles from the carrier, used ineach Example and Comparative Example, and the lane numbers in Westernblotting to be described later are shown in the following Table 15.

TABLE 15 Comparative Comparative Comparative Comparative Example 34Example 35 Example 10 Example 11 Example 12 Example 13 Method Obtainingmethod of the present Anti-CD63 antibody method Exosome-Human CD63invention Isolation/Detection Protein or antibody FLAG tag fusion typeAnti-CD63 antibody (H5C6) Anti-CD63 antibody to be immobilizedmouse-derived Tim-4 protein to carrier (Biotin-labeled SH-group) CarrierDynabeads M-270 Streptavidin Dynabeads M-270 Carboxylic Acid — Eluent 1%1 mM 1% 1 mM 1% 1 mM SDS EDTA SDS EDTA SDS EDTA Lane number in FIG. 9 12 3 4 5 6<Western Blotting>

Western blotting was carried out by the same method as in “(7) Westernblotting” of Examples 1 to 8, except for using “7.5 μL of eachsupernatant (eluate) obtained in Examples 34 to 35 and ComparativeExamples 10 to 13”, instead of “7.5 μL of each supernatant (eluate)obtained in Examples 1 to 8”.

<Results>

The results of Western blotting obtained are shown in FIG. 9. In FIG. 9,each lane is as follows:

Lane 1: Result of Example 34 (the results of the case where theextracellular membrane vesicles were obtained by the obtaining method ofthe present invention, and the 1% SDS aqueous solution was used as theeluent);

Lane 2: Result of Example 35 (the results of the case where theextracellular membrane vesicles were obtained by the obtaining method ofthe present invention, and the 1 mM EDTA aqueous solution was used asthe eluent);

Lane 3: Results of Comparative Example 10 (the results of the case wherethe extracellular membrane vesicles were obtained by the anti-CD63antibody method, and the 1% SDS aqueous solution was used as theeluent);

Lane 4: Results of Comparative Example 11 (the results of the case wherethe extracellular membrane vesicles were obtained by the anti-CD63antibody method, and the 1 mM EDTA aqueous solution was used as theeluent);

Lane 5: Result of Comparative Example 12 (the results of the case wherethe extracellular membrane vesicles were obtained by Exosome-Human CD63Isolation/Detection, and the 1% SDS aqueous solution was used as theeluent);

Lane 6: Result of Comparative Example 13 (the results of the case wherethe extracellular membrane vesicles were obtained by Exosome-Human CD63Isolation/Detection, and the 1 mM EDTA aqueous solution was used as theeluent).

As shown in FIG. 9, a band of CD63, which is one of exosome markers, wasobserved at the vicinity of 25 to 60 kDa in Examples 34 to 35, whereinobtaining of the extracellular membrane vesicles was carried out by theobtaining method of the present invention (Examples 34 to 35: lanes 1 to2).

On the other hand, as shown in FIG. 9, the band of CD63 was observedonly slightly at the vicinity of 25 to 60 kDa (Comparative Examples 10to 13: the lanes 3 to 6), in Comparative Examples 10 to 11 whereinobtaining of the extracellular membrane vesicles was carried out by theanti-CD63 antibody method, and in Comparative Examples 12 to 13 whereinobtaining of the extracellular membrane vesicles was carried out by theExosome-Human CD63 Isolation/Detection.

From the above, it has been revealed that more extracellular membranevesicles can be recovered, according to the obtaining method of thepresent invention, as compared with the method for obtaining theextracellular membrane vesicles by affinity of the surface antigenprotein and the antibody, using the antibody against the surface antigenprotein of the extracellular membrane vesicles in, such as the anti-CD63antibody method, or Exosome-Human CD63 Isolation/Detection, or the like.

Examples 36 to 38. Obtaining of Residual Extracellular Membrane Vesiclesfrom the Ultracentrifugal Separation-Treated Sample by Obtaining Methodof the Present Invention

As described below, the obtaining method of the present invention wascarried out for the sample from which the extracellular membranevesicles had been removed and obtained by the ultracentrifugalseparation method which is a conventional method (hereinafter it may beabbreviated as ultracentrifugal separation treatment, in some cases).

<(1) Removal of Extracellular Membrane Vesicles by UltracentrifugalSeparation Method>

FBS (15 mL, produced by Corning Inc.) was subjected to centrifugalseparation treatment (10,000×G, for 20 minutes) to separate impurities,and the supernatant was transferred to a new tube to obtain thecentrifugal separation treated FBS. Next, each 5 mL of the resultingcentrifugal separation treated FBS was subjected to ultracentrifugalseparation treatment (110,000×G, overnight) or ultracentrifugalseparation treatment (450,000×G, overnight), and the supernatant wastransferred to a new tube to obtain each 5 mL of the ultracentrifugalseparation-treated (110,000×G) FBS and ultracentrifugal separationtreated (450,000×G) FBS.

<(2) Washing of Beads>

The PBS solution (180 μL) containing the 10 μg/μL Dynabeads MyOneStreptavidin C1 (produced by Thermo Fisher Scientific Inc.) (containing1.8 mg of the Dynabeads MyOne Streptavidin C1) was dispensed into eachof three tubes. Next, each 1500 μL of PBS was added to the tube, andafter stirring, each tube was loaded on a magnet stand, the DynabeadsMyOne Streptavidin C1 was collected on the tube wall by using magneticforce, and the solution in the tube was discarded with a pipette(hereinafter it may be abbreviated as the washing operation, in somecases).

<(3) Immobilization of Fc Tag Fusion Type Mouse-Derived Tim-4 Protein onBeads>

The PBS solution (1500 μL) containing 3 μg of the SH-groupbiotin-labeled Fc tag fusion type mouse-derived Tim-4 protein was eachadded to 1.8 mg of the pellet-like Dynabeads MyOne Streptavidin C1 afterthe washing operation, and subjected to a reaction at 8° C. for 10minutes to obtain each 1500 μL of the PBS solution containing thecarrier on which the SH-group biotin-labeled Fc tag fusion type mTim-4protein was bound (mTim-4 carrier).

<(4) Obtaining of Extracellular Membrane Vesicles by Obtaining Method ofthe Present Invention>

The resulting mTim-4 carrier (1.8 mg) was subjected to the washingoperation each three times with 1500 μL of PBS to obtain the mTim-4carrier in a pellet state. Thereafter, each 5 mL of the centrifugalseparation treated FBS, the ultracentrifugal separation treated FBS(110,000×G) and the ultracentrifugal separation treated FBS (450,000×G)was dispensed into three 15 mL centrifuge tubes (manufactured by CorningInc.), then 1.8 mg of the mTim-4 carrier in a pellet state was added andsubjected to a reaction at 8° C. for 2 hours. After the reaction, the 15mL centrifuge tubes were loaded on a magnet stand, and the mTim-4carrier was collected on the tube wall using magnetic force, and eachsupernatant (each FBS) was recovered in each of new 15 mL centrifugetubes. The pellet-like mTim-4 carrier remained in the centrifuge tubewas each subjected to the washing operation with 3 mL of TBS-Tcontaining 2 mM CaCl₂ (Tris buffer, 0.0005% Tween 20, 2 mM CaCl₂), andthen each 1 mL of TBS-T containing 2 mM CaCl₂ was added to the mTim-4carrier in a pellet state and suspended, and the entire amount of thesuspension was transferred to the 1.5 mL tube, followed by subjecting tothe washing operation each twice.

Each 50 μL of the TBS solution containing 1 mM EDTA, as the eluent, wasadded to 1.8 mg of the pellet-like mTim-4 carrier, and they were mixedat room temperature for 10 seconds using a vortex mixer and spun down.Each of the 1.5 mL tubes was loaded on a magnet stand, and the mTim-4carrier was collected on the tube wall using magnetic force to recoverthe eluate. The TBS solution (50 μL) containing 1 mM EDTA was addedagain to recover the eluate in the same method using the mTim-4 carrierused above.

Thereafter, the beads, used when the eluate was recovered twice, wereagain added to each supernatant (each FBS) recovered in the 15-mLcentrifuge tube, and the operation for recovering the extracellularmembrane vesicles in each supernatant (each FBS) was repeated furthertwice by the same method as described above to obtain each eluate.

<(5) Western Blotting>

The resulting each eluate (100 μL) was ultrafiltrated with the VIVASPIN500 (MVCO 10,000) (manufactured by Sartorius AG), so as to attain 30 μL.Five μL of the 4×sample buffer solution (produced by Wako Pure ChemicalIndustries, Ltd.) was added to each 15 μL of the ultrafiltrated eluate,and incubated at 98° C. for 5 minutes to obtain each sample for Westernblotting. Each 20 μL of the sample for Western blotting was loaded onthe Super Sep Ace 5-20% gel (produced by Wako Pure Chemical IndustriesLtd.), and subjected to electrophoresis at 25 mA for 65 minutes. Theresulting electrophoresis gel was transcribed onto the PVDF membrane(produced by Millipore Corporation) at 1 mA/cm² for 60 minutes using thesemi-dry blotter and the discontinuous buffer (Anode buffer 1: 0.3 MTris/20% methanol, Anode buffer 2: 0.025 M Tris/20% methanol, Cathodebuffer: 0.025 M Tris/0.04 M aminocaproic acid/20% methanol). The 3%skimmed milk, diluted with PBS-T (PBS buffer, 0.1% Tween 20), was addedto the post-transcription PVDF membrane, and subjected to a reaction atroom temperature for 1 hour for blocking, and 2 mL of the anti-bovineCD9 antibody (produced by Novus Biologicals LLC, hereinafter it may beabbreviated as “the anti-bovine CD9 antibody”, in some cases), diluted1000-fold with PBS-T, was added and subjected to a reaction at roomtemperature for 1 hour. The PVDF membrane, after the reaction, waswashed three times with PBS-T, and the secondary antibody {anti-mouseIgG (H+L), rabbit, IgG fraction, peroxidase-conjugated antibody}(produced by Wako Pure Chemical Industries Ltd.), diluted 10,000-foldwith PBS-T, was subjected to a reaction at room temperature for 1 hour.After washing 5 times with PBS-T, the ImmunoStar Basic (produced by WakoPure Chemical Industries, Ltd.) was added to a membrane, which had beensubjected to a reaction with the anti-bovine CD9 antibody, and theImmunoStar Zeta (produced by Wako Pure Chemical Industries, Ltd.) wasadded to a membrane, which had been subjected to a reaction with theanti-Flotillin-2 antibody, and a luminescent signal was detected usingthe LAS-4000 (manufactured by GE Healthcare). It should be noted that,the anti-bovine CD9 antibody and the anti-Flotillin-2 antibody are theantibodies recognizing CD9 and Flotillin-2, which are marker proteins ofexosome, respectively.

<(6) Measurement of Average Particle Number and Average Particle Size>

Each 100 μL of the resulting eluate was treated with a centrifugalfilter (0.45 μm, PVDF) (manufactured by Millipore Corporation), anddiluted 5-fold with water, and then particles contained in the dilutedsolution were measured three times using a NanoSight LM 10 (manufacturedby NanoSight Ltd.), according to a manual of the NanoSight, and theaverage particle number (Particles) and the average particle size (MeanSize) were examined.

It should be noted that, the method for treating the samples, and themethod for obtaining the extracellular membrane vesicles remaining inthe sample, used in each Example, are shown in the following Table 16.

TABLE 16 Example 36 Example 37 Example 38 Treatment method of sampleUltracentrifugal separation — ◯ ◯ method (110000 × g) (450000 × g)(gravity in ultracentrifugal separation method) + Obtaining ofextracellular Obtaining method of the ◯ ◯ ◯ membrane vesicles presentinvention remaining in a sample by the method of the present inventionLane number in FIG. 10 1 2 3<Results>

The results of Western blotting obtained, and the measurement results ofaverage particle number and average particle size by the NanoSight areshown in FIG. 10 and Table 17, respectively. In FIG. 10, each lane showsthe following result:

Lane 1: Result of Example 36 (the results of the case where the samplewas centrifugal separation treated and obtaining of the extracellularmembrane vesicles was carried out by the obtaining method of the presentinvention);

Lane 2: Results of Example 37 (the results of the case where obtainingof the extracellular membrane vesicles was carried out by the obtainingmethod of the present invention, after subjecting the sample tocentrifugal separation treatment and ultracentrifugal separationtreatment at 110,000×G);

Lane 3: Results of Example 38 (the results of the case where obtainingof the extracellular membrane vesicles was carried out by the obtainingmethod of the present invention, after subjecting the sample tocentrifugal separation treatment and ultracentrifugal separationtreatment at 450,000×G).

From FIG. 10, it has been revealed that bands of CD9 and Flotillin-2which are the exosome markers were confirmed, and the extracellularmembrane vesicles contained in the sample could not be removed(obtained) completely by the ultracentrifugal separation treatment, anda large quantity of the extracellular membrane vesicles remained in thesample, also in the case where obtaining of the extracellular membranevesicles was carried out by the obtaining method of the presentinvention, after subjecting the sample to centrifugal separationtreatment, and ultracentrifugal separation treatment at 110,000×G or at450,000×G (Examples 37 to 38), (Examples 37 to 38: Lanes 2 to 3).Furthermore, it has been revealed that the extracellular membranevesicles remained in the sample, which were not removed (obtained)completely by these ultracentrifugal separation methods, can be obtained(removed) by the obtaining method of the present invention (Examples 37to 38: Lanes 2 to 3).

From Table 17, it was confirmed that size (particle size) of theextracellular membrane vesicles obtained was almost the same between thecase where obtaining of the extracellular membrane vesicles was carriedout by the obtaining method of the present invention, after subjectingthe sample to centrifugal separation treatment and ultracentrifugalseparation treatment at 110,000×G or at 450,000×G (Examples 37 to 38),and the case where obtaining of the extracellular membrane vesicles wascarried out by the obtaining method of the present invention, aftersubjecting the sample to centrifugal separation treatment (Example 36).Thus, it has been revealed that the extracellular membrane vesiclesremaining in the sample, which could not be removed completely by theultracentrifugal separation method, were not fragmented and was presentas particles.

TABLE 17 Particles Mean size (nm) Example 36 {circle around (1)}Untreated 2.04 × 10⁹ 155 Example 37 {circle around (2)} 110,000 × g 1.36× 10⁹ 162 Example 38 {circle around (3)} 450,000 × g 1.20 × 10⁹ 159

Examples 39 to 40. Obtaining of Remaining Extracellular MembraneVesicles by Obtaining Method of the Present Invention from Sample afterTreatment by Total Exosome Isolation (Polymer Precipitation)

As described below, the obtaining method of the present invention wascarried out for the sample, from which the extracellular membranevesicles contained had been removed (obtained) by Total ExosomeIsolation (polymer precipitation), which is a conventional method(hereinafter it may be abbreviated as “the polymer precipitationtreatment”, in some cases).

<(1) Removal of Extracellular Membrane Vesicles by Total ExosomeIsolation (Polymer Precipitation) Method>

FBS (10 mL, produced by Corning Inc.) was subjected to centrifugalseparation treatment (10,000×G, for 20 minutes) to separate impuritiesand obtain the centrifugal separation treated FBS. Subsequently, 1 mL ofthe Total Exosome Isolation (from serum) (produced by Thermo FisherScientific Inc.) was added to 5 mL of the resulting centrifugalseparation treated FBS, and allowed to stand under refrigeration for 30minutes, and centrifugal separation treated (10,000×G, for 20 minutes),then the supernatant was recovered to obtain 6 mL of FBS treated withthe Total Exosome Isolation.

<(2) Washing of Beads>

The PBS solution (180 μL) containing the 10 μg/μL Dynabeads MyOneStreptavidin C1 (produced by Thermo Fisher Scientific Inc.) (containing1.8 mg of the Dynabeads MyOne Streptavidin C1) was dispensed into eachof the two tubes. Next, 1500 μL of PBS was added to each of the tubes,and after stirring, each tube was loaded on a magnet stand, theDynabeads MyOne Streptavidin C1 was collected on the tube wall by usingmagnetic force, and the solution in the tube was discarded with apipette (hereinafter it may be abbreviated as the washing operation, insome cases).

<(3) Immobilization of Fc Tag Fusion Type Mouse-Derived Tim-4 Protein onBeads>

The PBS solution (1500 μL) containing 3 μg of the SH-groupbiotin-labeled Fc tag fusion type mouse-derived Tim-4 protein, preparedby the same method as in Examples 5 to 8, was added to 1.8 mg of theDynabeads MyOne Streptavidin C1, in a pellet state, after the washingoperation, and subjected to a reaction at 8° C. for 10 minutes to obtaineach 1500 μL of the PBS solution containing the carrier on which theSH-group biotin-labeled Fc tag fusion type mouse-derived Tim-4 proteinwas bound (the mTim-4 carrier).

<(4) Obtaining of Extracellular Membrane Vesicles by Obtaining Method ofthe Present Invention>

The resulting mTim-4 carrier (1.8 mg) was washed each three times with1500 μL of PBS to obtain the mTim-4 carrier in a pellet state.Thereafter, each 1.8 mg of the mTim-4 carrier in a pellet state wasadded to 5 mL of the centrifugal separation-treated FBS or 6 mL of theTotal Exosome Isolation-treated FBS which had been dispensed into 15 mLcentrifuge tubes (produced by Corning Inc.), and subjected to a reactionat 8° C. for 2 hours.

After the reaction, each of the 15 mL centrifuge tubes were loaded on amagnet stand, and the mouse-derived Tim-4 carrier was collected on thetube wall by magnetic force to recover each supernatant (each FBS) innew 15 mL centrifuge tubes. The pellet-like mTim-4 carrier remained inthe centrifuge tubes was subjected to the washing operation each with 3mL of TBS-T containing 2 mM CaCl₂ (Tris buffer, 0.0005% Tween 20, 2 mMCaCl₂), and then each 1 mL of TBS-T containing 2 mM CaCl₂ was added tothe pellet-like mTim-4 carrier and suspended, and after the entireamount of the suspension was transferred to the 1.5 mL tube, the washingoperation was each carried out twice.

The TBS solution (50 μL) containing 1 mM EDTA, as the eluent, was addedto each 1.8 mg of the mTim-4 carrier in a pellet state, and then theywere mixed using a vortex mixer at room temperature for 10 seconds andspun down. Each 1.5 mL tube was loaded on a magnet stand, and the mTim-4carrier was collected on the tube wall using magnetic force to recoverthe eluate. The TBS solution (50 μL) containing 1 mM EDTA was addedagain to recover the eluate in the same method using the mTim-4 carrierused above.

Thereafter, the mTim-4 carrier (beads) used in recovering the eluatetwice was added again to each supernatant (each FBS) recovered in the 15mL-centrifuge tube, and by the same method as described above, theprocedure of recovering the extracellular membrane vesicles in eachsupernatant (each FBS) was repeated further twice to obtain each eluate.

<(5) Western Blotting>

Each 300 μL of the resulting eluate was ultrafiltrated with the VIVASPIN500 (MVCO 10,000) (produced by Sartorius AG), so as to attain 30 μL.Each 10 μL of the 4× sample buffer (produced by Wako Pure ChemicalIndustries, Ltd.) was added to 30 μL of each ultrafiltrated eluate, andincubated at 98° C. for 5 minutes to obtain each sample for Westernblotting. Each 20 μL of the sample for Western blotting was loaded ateach 2 places on the Super Sep Ace 5-20% gel (produced by Wako PureChemical Industries, Ltd.) and subjected to electrophoresis at 25 mA for65 minutes. The resulting electrophoresis gel was transcribed onto thePVDF membrane (produced by Millipore Corporation) at 1 mA/cm² for 60minutes using the semi-dry blotter and the discontinuous buffer (Anodebuffer 1: 0.3 M Tris/20% methanol, Anode buffer 2: 0.025 M Tris/20%methanol, Cathode buffer: 0.025 M Tris/0.04 M aminocaproic acid/20%methanol). The 3% skimmed milk, diluted with PBS-T (PBS buffer, 0.1%Tween 20), was added to the post-transcription PVDF membrane, andsubjected to a reaction at room temperature for 1 hour for blocking, and2 mL of the anti-bovine CD9 antibody (produced by Novus Biologicals LLC,hereinafter it may be abbreviated as “the anti-bovine CD9 antibody”, insome cases), diluted 1000-fold with PBS-T, or the anti-human Flotillin-2antibody (produced by BD Biosciences, hereinafter it may be abbreviatedas “the anti-human Flotillin-2 antibody”, in some cases), diluted250-fold with PBS-T, was added and subjected to a reaction at roomtemperature for 1 hour. After the reaction, the PVDF membrane was washedthree times with PBS-T, and the secondary antibody {anti-mouse IgG(H+L), rabbit, IgG fraction, peroxidase-conjugated antibody} (producedby Wako Pure Chemical Industries Ltd.), diluted 10,000-fold with PBS-T,was subjected to a reaction at room temperature for 1 hour. Afterwashing 5 times with PBS-T, the ImmunoStar Basic (produced by Wako PureChemical Industries, Ltd.) was added to a membrane, which had beensubjected to a reaction with the anti-bovine CD9 antibody, and theImmunoStar Zeta (produced by Wako Pure Chemical Industries, Ltd.) wasadded to a membrane, which had been subjected to a reaction with theanti-Flotillin-2 antibody, and a luminescent signal was detected usingthe LAS-4000 (manufactured by GE Healthcare). It should be noted that,the anti-bovine CD9 antibody is an antibody against CD9 which is one ofthe marker proteins of exosome, and the anti-human Flotillin-2 antibodyis an antibody against Flotillin-2 which is one of the marker proteinsof exosome.

It should be noted that, the method for treating the sample, and themethods of obtaining the extracellular membrane vesicles remaining inthe sample, used in each Example, and the lane number in Westernblotting are shown in the following Table 18.

TABLE 18 Exam- Exam- ple 39 ple 40 Treatment method of Total Exosome — ◯sample Isolation (PEG precipitation) method + Obtaining of extracellularObtaining method of ◯ ◯ membrane vesicles remaining the present in asample by the invention method of the present invention Lane number inFIG. 11 1 2<Results>

The results of Western blotting obtained are shown in FIG. 11. In FIG.11, each lane represents the following result.

Lane 1: Results of Example 39 (the results of the case where obtainingof the extracellular membrane vesicles was carried out by the obtainingmethod of the present invention, after subjecting the sample tocentrifugal separation treatment);

Lane 2: Results of Example 40 (the results of the case where obtainingof the extracellular membrane vesicles was carried out by the obtainingmethod of the present invention, after subjecting the sample tocentrifugal separation treatment and polymer precipitation treatment).

From FIG. 11, it has been revealed that also in the case where obtainingof the extracellular membrane vesicles were carried out by the obtainingmethod of the present invention, after subjecting the sample tocentrifugal separation treatment, and the polymer precipitation (Example40), the extracellular membrane vesicles contained in the sample couldnot be removed (obtained) completely by the Total Exosome Isolation(polymer precipitation) method, and a large quantity of theextracellular membrane vesicles remained in the sample, from the factthat bands of CD9 and Flotillin-2, which are the exosome markers, wereconfirmed (Example 40: Lane 2).

Furthermore, it has been revealed that the extracellular membranevesicles remained in the sample that were not removed (obtained)completely by the Total Exosome Isolation (polymer precipitation) methodcan be obtained by the obtaining method of the present invention(Example 40: Lane 2).

Examples 41 to 47. Obtaining (Removal) of Extracellular MembraneVesicles from Sample by Removing Method of the Present Invention and/orConventional Method (Ultracentrifugation Method, Total Exosome Isolation(Polymer Precipitation) Method)

As described below, the extracellular membrane vesicles were obtained(removed) from the sample by the removing method of the presentinvention and/or the conventional method (ultracentrifugation method,Total Exosome Isolation (polymer precipitation) method).

<(1) Removal of Extracellular Membrane Vesicles by UltracentrifugalSeparation Method>

FBS (28 mL, produced by Corning Inc.) was subjected to centrifugalseparation treatment (10,000×G, for 20 minutes) to separate impuritiesand obtain centrifugal separation treated FBS (hereinafter it may beabbreviated as “the removed sample-1”, in some cases). Subsequently, 12mL of the resulting centrifugal separation treated FBS (“the removedsample-1”) was subjected to ultracentrifugal separation treatment(110,000×G, overnight) to obtain ultracentrifugal separation treated FBS(110,000×G) (hereinafter it may be abbreviated as “the removedsample-2”, in some cases).

<(2) Removal of Extracellular Membrane Vesicles by Total ExosomeIsolation (Polymer Precipitation) Method>

Subsequently, 1.6 mL of Total Exosome Isolation (from serum) (producedby Thermo Fisher Scientific Inc.) was added to 8 mL of the centrifugalseparation treated FBS (“the removed sample-1”), and allowed to standunder refrigeration for 30 minutes, and centrifugal separation treated(10,000×G, for 20 minutes), then each supernatant was recovered toobtain 9.6 mL of Total Exosome Isolation treated FBS (hereinafter it maybe abbreviated as “the removed sample-3”, in some cases).

In addition, 0.8 mL of Total Exosome Isolation was added to 4 mL of theultracentrifugal separation treated (110,000×G) FBS (“the removedSample-2”), and allowed to stand under refrigeration for 30 minutes, andcentrifugal separation treated (10,000×G, 20 minutes) to recover eachsupernatant and obtain 4.8 mL of ultracentrifugal separation treated(110,000×G)/Total Exosome Isolation treated FBS (hereinafter it may beabbreviated as “the removed sample-4”, in some cases).

<(3) Washing of Beads>

The PBS solution (240 μL) containing the 10 μg/μL Dynabeads MyOneStreptavidin C1 (produced by Thermo Fisher Scientific Inc.) (containing2.4 mg of the Dynabeads MyOne Streptavidin C1) was dispensed into eachof three tubes. Next, 2000 μL of PBS was added to each of the tubes, andafter stirring, each tube was loaded on a magnet stand, the DynabeadsMyOne Streptavidin C1 was collected on the tube wall using magneticforce, and the solution in the tube was discarded with a pipette(hereinafter it may be abbreviated as “the washing operation”, in somecases).

<(4) Immobilization of Fc Tag Fusion Type Mouse-Derived Tim-4 Protein onBeads>

The PBS solution (2000 μL) containing 4 μg of the SH-groupbiotin-labeled Fc tag fusion type mouse-derived Tim-4 protein was addedto each 2.4 mg of the Dynabeads MyOne Streptavidin C1 in a pellet state,after the washing operation, and subjected to a reaction at 8° C. for 10minutes to obtain each 2000 μL of the PBS solution containing thecarrier on which the SH-group biotin-labeled Fc tag fusion typemouse-derived Tim-4 protein was bound (the mTim-4 carrier).

<(5) Removal of Extracellular Membrane Vesicles>

The resulting 2.4 mg of the mTim-4 carrier was subjected to the washingoperation three times with each 2000 μL of PBS to obtain the mTim-4carrier in a pellet state.

Thereafter, each 4 mL of centrifugal separation treated FBS (“theremoved sample-1”), 4 mL of ultracentrifugal separation treated(110,000×G) FBS (“the removed sample-2”), or 4.8 mL of Total ExosomeIsolation treated FBS (“the removed sample-3”) was dispensed into eachtube and subjected to a reaction at 8° C. for 1 hour. After thereaction, each of the tubes was loaded on a magnet stand, and the mTim-4carrier was collected on the tube wall using magnetic force to recoversupernatant (each FBS) in a new tube.

The mTim-4 carrier in a pellet state remaining in the tube was subjectedto the washing operation three times with each 4 mL of TBS-T (Trisbuffer, 0.0005% Tween 20), and then each supernatant (each FBS) wasadded again, and each of the operations from the reaction to washing wasrepeated five times in total. After the fifth time reaction, eachsupernatant (each FBS) was transferred to a new tube and centrifugalseparation treated (10,000×G, for 20 minutes), then the supernatant wasrecovered to obtain 4 mL of ultracentrifugal separation treated(110,000×G)/Tim-4 treated FBS (hereinafter it may be abbreviated as “theremoved sample-5”, in some cases), 4.8 mL of Total Exosome Isolationtreated/Tim-4 treated FBS (hereinafter it may be abbreviated as “theremoved sample-6”, in some cases), and 4 mL of the Tim-4 treated FBS(hereinafter it may be abbreviated as “the removed sample-7”, in somecases).

<(6) Obtaining of Extracellular Membrane Vesicles by Obtaining Method ofthe Present Invention>

Seven 0.6 mg mTim-4 carriers were newly prepared by the same way as theabove method, and each was subjected to the washing operation. Theremoved sample described in the following Table 19 was added to 0.6 mgof the mTim-4 carrier in a pellet state, in each addition amount to themouse-derived Tim-4 carrier of the removed sample, and each wassubjected to a reaction at 8° C. for 3 hours.

TABLE 19 Example Example Example Example Example Example Example 41 4243 44 45 46 47 Name of sample obtained in each Example Removed RemovedRemoved Removed Removed Removed Removed sample-5 sample-6 sample-4sample-2 sample-3 sample-7 sample-1 Centrifugal separation treatment ◯ ◯◯ ◯ ◯ ◯ ◯ Treatment Ultracentrifugal separation ◯ — ◯ ◯ — — — method ofMethod (gravity in (110,000 × g) (110,000 × g) (110,000 × g) sampleultracentrifugation method) Total Exosome Isolation (PEG — ◯ ◯ — ◯ — —precipitation) method Removing method of ◯ ◯ — — — ◯ — the presentinvention Addition amount of removed sample to 1 mL 1.2 mL 1.2 mL 1 mL1.2 mL 1 mL 1 mL mouse-derived Tim-4 carrier

After the reaction, each 1.5 mL tube was loaded on a magnet stand, andthe mTim-4 carrier was collected on the tube wall by magnetic force, andeach supernatant (each FBS) was recovered to obtain the mTim-4 carrierin a pellet state.

The resulting mTim-4 carrier in a pellet state was subjected to thewashing operation three times with each 1 mL of TBS-T containing 2 mMCaCl₂ (Tris buffer, 0.0005% Tween 20, 2 mM CaCl₂), and then each 50 μLof the TBS solution containing 1 mM EDTA, as the eluent, was added toeach 0.6 mg of the mTim-4 carrier in a pellet state, and they were mixedusing a vortex mixer at room temperature for 10 seconds, and spun down.Each 1.5 mL tube was loaded on a magnet stand, and the mTim-4 carrierwas collected on the tube wall using magnetic force to recover eacheluate. The TBS solution (50 μL) containing 1 mM EDTA was added again torecover each eluate in the same method.

<(7) Western Blotting>

In the resulting eluate, 100 μL was ultrafiltrated, so as to attain 30μL with the VIVASPIN 500 (MVCO 10,000) (produced by Sartorius AG). Toeach 30 μL of the ultrafiltrated eluate, 10 μL of the 4× sample buffer(produced by Wako Pure Chemical Industries, Ltd.) was added andincubated at 98° C. for 5 minutes to obtain each sample for Westernblotting. The sample for Western blotting (each 20 μL) was loaded, bytwo places, on the Super Sep Ace 5-20% gel (produced by Wako PureChemical Industries, Ltd.), and subjected to electrophoresis at 25 mAfor 65 minutes. The resulting electrophoresis gel was transcribed ontothe PVDF membrane (produced by Millipore Corporation) under 1 mA/cm² for60 minutes using the semi-dry blotter and the discontinuous buffer(Anode buffer 1: 0.3 M Tris/20% methanol, Anode buffer 2: 0.025 MTris/20% methanol, Cathode buffer: 0.025 M Tris/0.04 M aminocaproicacid/20% methanol). To the PVDF membrane after transcription, the 3%skimmed milk diluted with PBS-T (the PBS buffer, 0.1% Tween 20) wasadded and subjected to a reaction at room temperature for 1 hour forblocking, and 2 mL of the anti-bovine CD9 antibody (produced by NovusBiologicals LLC, hereinafter it may be abbreviated as “the anti-bovineCD9 antibody”, in some cases) diluted 1000-fold with PBS-T, or theanti-human Flotillin-2 antibody (produced by BD Biosciences, hereinafterit may be abbreviated as “the anti-human Flotillin-2 antibody”, in somecases), diluted 250-fold with PBS-T, was subjected to a reaction at roomtemperature for 1 hour. After the reaction, the PVDF membrane was washedthree times with PBS-T, and the secondary antibody {anti-mouse IgG(H+L), rabbit, IgG fraction, peroxidase-conjugated antibody} (producedby Wako Pure Chemical Industries Ltd.), diluted 10,000-fold with PBS-T,was subjected to a reaction at room temperature for 1 hour. Afterwashing five times with PBS-T, the ImmunoStar Basic (produced by WakoPure Chemical Industries, Ltd.) was added to the membrane after beingsubjected to a reaction with the anti-bovine CD9 antibody, and theImmunoStar Zeta (produced by Wako Pure Chemical Industries, Ltd.) wasadded to the membrane after being subjected to a reaction with theanti-Flotillin-2 antibody, and a luminescent signal was detected usingthe LAS-4000 (manufactured by GE Healthcare). It should be noted thatthe anti-bovine CD9 antibody is an antibody against CD9, which is one ofthe marker proteins of exosome, and the anti-human Flotillin-2 antibodyis an antibody against Flotillin-2, which is one of the marker proteinsof exosome.

It should be noted that, the method for treating the sample, the methodfor obtaining (removing) the extracellular membrane vesicles remainingin the sample, used in each Example, and the lane numbers in Westernblotting to be described later are shown in the following Table 20.

TABLE 20 Example Example Example Example Example Example Example 41 4243 44 45 46 47 Name of sample obtained in each Example Removed RemovedRemoved Removed Removed Removed Removed sample-5 sample-6 sample-4sample-2 sample-3 sample-7 sample-1 Centrifugal separation treatment ◯ ◯◯ ◯ ◯ ◯ ◯ Treatment Ultracentrifugal separation ◯ — ◯ ◯ — — — method ofmethod (gravity in (110,000 × g) (110,000 × g) (110,000 × g) sampleultracentrifugation method) Total Exosome Isolation (PEG — ◯ ◯ — ◯ — —precipitation) method Removing method of ◯ ◯ — — — ◯ — the presentinvention Addition amount of removed sample to 1 mL 1.2 mL 1.2 mL 1 mL1.2 mL 1 mL 1 mL mouse-derived Tim-4 carrier + Obtaining ofextracellular membrane vesicles ◯ ◯ ◯ ◯ ◯ ◯ ◯ remaining in the sample bythe obtaining method of the present invention Lane number in FIG. 12 1 23 4 5 6 7<Results>

The results of Western blotting obtained are shown in FIG. 12. In FIG.12, each lane shows the following result.

Lane 1: Results of Example 41 (the results of the case where obtainingof the extracellular membrane vesicles was carried out by the obtainingmethod of the present invention, after subjecting the sample tocentrifugal separation treatment, ultracentrifugal separation treatment,and further treatment with the removing method of the presentinvention);

Lane 2: Results of Example 42 (the results of the case where obtainingof the extracellular membrane vesicles was carried out by the obtainingmethod of the present invention, after subjecting the sample tocentrifugal separation treatment, polymer precipitation treatment, andfurther treatment with the removing method of the present invention);

Lane 3: Results of Example 43 (the results of the case where obtainingof the extracellular membrane vesicles was carried out by the obtainingmethod of the present invention, after subjecting the sample tocentrifugal separation treatment, ultracentrifugal separation treatment,and further polymer precipitation treatment);

Lane 4: Results of Example 44 (the results of the case where obtainingof the extracellular membrane vesicles was carried out by the obtainingmethod of the present invention, after subjecting the sample tocentrifugal separation treatment, and ultracentrifugal separationtreatment);

Lane 5: Results of Example 45 (the results of the case where obtainingof the extracellular membrane vesicles was carried out by the obtainingmethod of the present invention, after subjecting the sample tocentrifugal separation treatment, and polymer precipitation treatment);

Lane 6: Results of Example 46 (the results of the case where obtainingof the extracellular membrane vesicles was carried out by the obtainingmethod of the present invention, after subjecting the sample tocentrifugal separation treatment, and treatment with the removing methodof the present invention);

Lane 7: Results of Example 47 (the results of the case where obtainingof the extracellular membrane vesicles was carried out by the obtainingmethod of the present invention, after subjecting the sample tocentrifugal separation treatment).

From FIG. 12, it has been revealed that the extracellular membranevesicles contained in the sample cannot be removed completely, and alarge quantity of the extracellular membrane vesicles remain in thesample, from the fact that the bands of exosome markers CD9 andFlotillin-2 are observed, in the ultracentrifugation method and TotalExosome Isolation (polymer precipitation) method, which are theconventional methods (Examples 44 to 45: lanes 4 to 5). In addition, ithas been revealed that the extracellular membrane vesicles contained inthe sample cannot be removed sufficiently, and resulted in to remain,from the fact that the bands of CD9 and Flotillin-2 are observed, evenif these conventional methods are combined (Example 43: lane 3). On theother hand, it has been revealed that the extracellular membranevesicles in the sample, which cannot be removed only by the conventionalmethods, can be removed (obtained) by combining the ultracentrifugationmethod or Total Exosome Isolation (polymer precipitation) method, whichare the conventional methods, with the removing method of the presentinvention, from the fact that the bands of CD9 and Flotillin-2 are notconfirmed (Examples 41 to 42: lanes 1-2). Furthermore, it has beenrevealed that the extracellular membrane vesicles in the sample can beremoved sufficiently, even with the method of the present inventionalone (Example 46: lane 6). In addition, it has been suggested that theviruses can also be removed similarly as the extracellular membranevesicles, according to the removing (obtaining) method of the presentinvention.

Examples 48 to 55. Reutilization of Tim Carrier of the Present Inventionin Removing Method of the Present Invention

The removing method of the present invention was repeated using the Timcarrier of the present invention as follows.

<(1) Preparation of Sample>

One mL of FBS (produced by Corning Inc.) was subjected to centrifugalseparation treatment (10,000×G, for 20 minutes) to separate impuritiesto obtain 1 mL of the centrifugal separation treated FBS.

<(2) Washing of Beads>

The PBS solution (60 μL) containing the 10 μg/μL Dynabeads MyOneStreptavidin C1 (produced by Thermo Fisher Scientific Inc.) (containing0.6 mg of the Dynabeads MyOne Streptavidin C1) was dispensed into the1.5 mL tube (manufactured by BM Equipment Co., Ltd.). Next, 500 μL ofPBS was added to each 1.5 mL tube, and after stirring, the 1.5 mL tubewas loaded on a magnet stand, the Dynabeads MyOne Streptavidin C1 wascollected on the tube wall using magnetic force, and the solution in the1.5 mL tube was discarded with a pipette (hereinafter it may beabbreviated as the washing operation, in some cases).

<(3) Immobilization of Fc Tag Fusion Type Mouse-Derived Tim-4 Protein onBeads>

The PBS solution (500 μL) containing 1 μg of the SH-group biotin-labeledFc tag fusion type mouse-derived Tim-4 protein was added to 0.6 mg ofthe Dynabeads MyOne Streptavidin C1, in a pellet state, after thewashing operation, and subjected to a reaction at 8° C. for 10 minutesto obtain 500 μL of the PBS solution containing the carrier on which theSH-group biotin-labeled Fc tag fusion type mouse-derived Tim-4 proteinwas bound (the mTim-4 carrier).

<(4) Removal of Extracellular Membrane Vesicles by Removing Method ofthe Present Invention>

The 1.5 mL tube was loaded on a magnet stand and the mTim-4 carrier wascollected on the tube wall using magnetic force, and the solution in the1.5 mL tube was discarded with a pipette, and then the mTim-4 carrierwas subjected to the washing operation three times with each 500 μL ofPBS to obtain the mTim-4 carrier in a pellet state. Thereafter, 1 mL ofthe centrifugal separation treated FBS was added to the mTim-4 carrierin a pellet state, and subjected to a reaction at 8° C. for 1 hour.

After the reaction, the 1.5 mL tube was loaded on a magnet stand, themTim-4 carrier was collected on the tube wall using magnetic force torecover the supernatant (FBS) in a new 1.5 mL tube (referred to assupernatant-1). The mTim-4 carrier in a pellet state was subjected tothe washing operation three times with 500 μL of TBS-T containing 2 mMCaCl₂ (Tris buffer, 0.0005% Tween 20, 2 mM CaCl₂).

The TBS solution (50 μL) containing 1 mM EDTA as the eluent was added to0.6 mg of each mTim-4 carrier in a pellet state, and then they weremixed using a vortex mixer at room temperature for 10 seconds, and spundown. The 1.5 mL tube was loaded on a magnet stand, and the mTim-4carrier was collected to the tube wall using a magnetic force to recoverthe eluate (referred to as the eluate-1). The TBS solution (50 μL)containing 1 mM EDTA was added again to the mTim-4 carrier to recoverthe eluate by the same method (referred to as eluate-2). The resultingeluate-1 and eluate-2 were mixed to obtain a removed sample-8.

The supernatant-1 was added again to the mTim-4 carrier after theelution operation (after the elution operation to obtain the eluate-2)and the operation of recovering the extracellular membrane vesicles inthe supernatant-1 was repeated eight times in total to obtain eacheluate.

The resulting removed samples and the lane numbers in Western blottingto be described later are as shown in the following Table 21.

TABLE 21 Example 48 Example 49 Example 50 Example 51 Example 52 Example53 Example 54 Example 55 Name of sample Removed Removed Removed RemovedRemoved Removed Removed Removed sample 8 sample 9 sample 10 sample 11sample 12 sample 13 sample 14 sample 15 Mouse-derived Tim-4 carrier and0 1 2 3 4 5 6 7 number of times (unused) sample reutilized Lane numbersin FIG. 13 1 2 3 4 5 6 7 8<(5) Western Blotting>

Each 100 μL of the resulting eluate was ultrafiltrated, so as to attain30 μL with the VIVASPIN 500 (MVCO 10,000) (manufactured by SartoriusAG). To each 15 μL of the ultrafiltrated eluate, 5 μL of the 4× samplebuffer (produced by Wako Pure Chemical Industries, Ltd.) was added andincubated at 98° C. for 5 minutes to obtain each sample for Westernblotting. Each 20 μL of the sample for Western blotting was loaded onthe Super Sep Ace 5-20% gel (produced by Wako Pure Chemical Industries,Ltd.) and subjected to electrophoresis at 25 mA for 65 minutes. Theresulting electrophoresis gel was transcribed onto the PVDF membrane(produced by Millipore Corporation) at 1 mA/cm² for 60 minutes using thesemi-dry blotter and the discontinuous buffer (Anode buffer 1: 0.3 MTris/20% methanol, Anode buffer 2: 0.025 M Tris/20% methanol, Cathodebuffer: 0.025 M Tris/0.04 M aminocaproic acid/20% methanol). The 3%skimmed milk, diluted with PBS-T (PBS buffer, 0.1% Tween 20), was addedto the post-transcription PVDF membrane, and subjected to a reaction atroom temperature for 1 hour for blocking, and 2 mL of the anti-bovineCD9 antibody (produced by Novus Biologicals LLC, hereinafter it may beabbreviated as “the anti-bovine CD9 antibody”, in some cases), diluted1,000-fold with PBS-T, was subjected to a reaction at room temperaturefor 1 hour. The PVDF membrane after the reaction was washed three timeswith PBS-T, and the secondary antibody {anti-mouse IgG (H+L), rabbit,IgG fraction, peroxidase-conjugated antibody} (produced by Wako PureChemical Industries Ltd.), diluted 10,000-fold with PBS-T, was subjectedto a reaction at room temperature for 1 hour. After washing five timeswith PBS-T, the ImmunoStar Zeta (produced by Wako Pure ChemicalIndustries, Ltd.) was added, and a luminescent signal was detected usingthe LAS-4000 (manufactured by GE Healthcare). It should be noted that,the anti-bovine CD9 antibody is an antibody against CD9, which is one ofthe marker proteins of exosome.

<Results>

The results of Western blotting obtained are shown in FIG. 13. In FIG.13, each lane shows the following result.

Lane 1: Results of Example 48 (the results of the case where theextracellular membrane vesicles in the sample were removed by theobtaining/removing method of the present invention using the unusedTim-4 carrier of the present invention);

Lane 2: Results of Example 49 (the results of the case where theextracellular membrane vesicles in the sample used once were removed bythe obtaining/removing method of the present invention using the Tim-4carrier of the present invention used once);

Lane 3: Results of Example 50 (the results of the case where theextracellular membrane vesicles in the sample used twice were removed bythe obtaining/removing method of the present invention using the Tim-4carrier of the present invention used twice);

Lane 4: Results of Example 51 (the results of the case where theextracellular membrane vesicles in the sample used 3 times were removedby the obtaining/removing method of the present invention using theTim-4 carrier of the present invention used 3 times);

Lane 5: Results of Example 52 (the results of the case where theextracellular membrane vesicles in the sample used 4 times were removedby the obtaining/removing method of the present invention using theTim-4 carrier of the present invention used 4 times);

Lane 6: Results of Example 53 (the results of the case where theextracellular membrane vesicles in the sample used 5 times were removedby the obtaining/removing method of the present invention using theTim-4 carrier of the present invention used 5 times);

Lane 7: Results of Example 54 (the results of the case where theextracellular membrane vesicles in the sample used 6 times were removedby the obtaining/removing method of the present invention using theTim-4 carrier of the present invention used 6 times);

Lane 8: Results of Example 55 (the results of the case where theextracellular membrane vesicles in the sample used 7 times were removedby the obtaining/removing method of the present invention using theTim-4 carrier of the present invention used 7 times).

From FIG. 13, it has been revealed that the extracellular membranevesicles can be removed more reliably, by utilizing the Tim-4 carrier ofthe present invention repeatedly, from the fact that when the removaloperation is carried out by repeatedly using the Tim-4 carrier of thepresent invention, amount of exosome recovered from the sampledecreases. In addition, it has been revealed that the Tim-4 carrier ofthe present invention is reusable, by separating the extracellularmembrane vesicles from the complex of the Tim-4 protein bound to thecarrier and the extracellular membrane vesicle in the sample, usingEDTA, which is the calcium ion chelating agent.

Examples 56 to 67, Comparative Examples 14 to 15. Obtaining ofExtracellular Membrane Vesicles by Carrier on which Tim Family Proteinis Immobilized

As described below, obtaining of the extracellular membrane vesiclespertaining to the present invention was carried out by using the carrierin which the Tim-1 protein, the Tim-3 protein, or the Tim-4 protein,which is the Tim family, was each immobilized on the beads.

<(1) Preparation of Calcium Ion-Containing Culture Supernatant (StockSolution)>

The human chronic myelogenous leukemia cell strain K562, 1×10⁷ cells,secreting the extracellular membrane vesicles, was cultured for threedays under condition at 37° C. and 5% CO₂, using 80 mL of the X-VIVO15medium (produced by Lonza AG), then the cells were precipitated bycentrifugal separation treatment (300×G, for 5 minutes) to remove thesupernatant. The precipitated cells were suspended in 60 mL of theX-VIVO 15 medium containing 10 μM monensin sodium (produced by MPBiomedicals Co., Ltd) and cultured for 24 hours under condition at 37°C. and 5% CO₂.

Thereafter, the culture solution was subjected to centrifugal separationtreatment (300×G, for 5 minutes) to recover the culture supernatant. Therecovered culture supernatant (60 mL) was further subjected tocentrifugal separation treatment three times (the first time: 300×G, for3 minutes, the second time: 1,200×G, for 20 minutes, the third time:10,000×G, for 20 minutes) to separate impurities to obtain thesupernatant (hereinafter it may be abbreviated as “the culturesupernatant (stock solution)”, in some cases).

In addition, CaCl₂ was added to the resulting culture supernatant stocksolution, so as to attain the final concentration of 2 mM to obtain asample of K562 cell culture supernatant concentrated solution containing2 mM CaCl₂ (hereinafter it may be abbreviated as “the calciumion-containing culture supernatant (stock solution)”, in some cases).

<(2) Washing of Beads>

The PBS-T solution (20 μL) containing the 30 μg/μL Dynabeads Protein G(produced by Thermo Fisher Scientific Inc.) (containing 0.6 mg of theDynabeads Protein G) was dispensed into seven 1.5 mL tubes (manufacturedby BM Equipment Co., Ltd.), and each subjected to the washing operation.

<(3) Immobilization of Fc Tag Fusion Type Tim Protein on Beads>

Each 200 μL of the TBS solution containing 1 μg of the Fc tag fusiontype mouse-derived Tim-1 protein, the Fc tag fusion type mouse-derivedTim-3 protein, the Fc tag fusion type mouse-derived Tim-4 protein, theFc tag fusion type human-derived Tim-1 protein, the Fc tag fusion typehuman-derived Tim-3 protein, or the Fc tag fusion type human-derivedTim-4 protein (all produced by Wako Pure Chemical Industries, Ltd.) wasadded to the 1.5 mL tubes containing 0.6 mg of Dynabeads Protein Grespectively, and subjected to a reaction at 8° C. for 1 hour to obtaineach of the carrier on which the Fc tag fusion type mTim-1 protein wasbound, the carrier on which the Fc tag fusion type mTim-3 protein wasbound, the carrier on which the Fc tag fusion type mTim-4 protein-boundcarrier, the carrier on which the Fc tag fusion type hTim-1 protein wasbound, the carrier on which the Fc tag fusion type hTim-3 protein wasbound, and the carrier on which the Fc tag fusion type hTim-4 proteinwas bound. It should be noted that, the resulting these six types ofcarriers are shown in the following Table 22. They may be abbreviatedcollectively as “the Tim protein-bound carrier”, in some cases.

TABLE 22 1 2 3 4 5 6 Tim protein- Carrier Dynabeads Protein G beadsbound carrier Fc tag fusion type Human- Mouse- Human- Mouse- Human-Mouse- Tim protein derived derived derived derived derived derived Tim-1Tim-1 Tim-3 Tim-3 Tim-4 Tim-4<(4) Obtaining of Extracellular Membrane Vesicles by Method of thePresent Invention>

The resulting 6 types of the Tim protein-bound carriers above were eachsubjected to the washing operation three times with 500 μL of TBS-T(Tris buffer, 0.1% Tween 20) and twice with 500 μL of TBS to obtain theTim protein-bound carriers in a pellet state. Subsequently, 1 mL of thecalcium ion-containing culture supernatant (stock solution) prepared inthe (1) was added to each of the Tim protein-bound carriers in a pelletstate, and the Dynabeads Protein G which had been subjected to only thewashing operation (hereinafter it may be abbreviated as “the Tim proteinnon-bound carrier”, in some cases), and each subjected to a reaction at8° C. for 3 hours.

Thereafter, the Tim protein-bound carrier or the Tim protein non-boundcarrier, after the reaction, was subjected to the washing operationthree times with each 500 μL of the calcium ion-containing TBS-T (Trisbuffer, 0.0005% Tween 20, 2 mM CaCl₂). At the third time of the washingoperation, 500 μL of the CaCl₂-containing TBS-T solution containing theTim protein-bound carrier or the Tim protein non-bound carrier wasdispensed into each of the two 1.5 mL tubes, by each 250 μL aliquot, andafter the tube was loaded on a magnetic stand, the washing solution wasremoved.

50 μL of 1% SDS aqueous solution or the TBS solution (25 μL) containing1 mM EDTA, as the eluent, was added to each of 0.3 mg of the pellet-likeTim protein-bound carrier, or the Tim protein non-bound carrier, andthey were mixed using a vortex mixer at room temperature for 10 secondsand spun down. The TBS solution (25 μL) containing 1 mM EDTA was addedagain to the tube to which EDTA had been added and the same operationwas carried out to obtain each supernatant (eluate).

<(5) Western Blotting>

The 4×sample buffer (3.75 μL, produced by Wako Pure Chemical Industries,Ltd.) was added to 11.25 μL of the resulting each supernatant (eacheluate) in the (4), and incubated at 95° C. for 3 minutes to obtain eachsample for Western blotting. Each 15 μL of the sample for Westernblotting was loaded on the Super Sep Ace 5-20% gel (produced by WakoPure Chemical Industries, Ltd.), and subjected to electrophoresis at 30mA for 60 minutes. The resulting electrophoresis gel was transcribedonto the PVDF membrane (produced by Millipore Corporation) at 1 mA/cm²for 60 minutes using the semi-dry blotter and the discontinuous buffer(Anode buffer 1: 0.3 M Tris/20% methanol, Anode buffer 2: 0.025 MTris/20% methanol, Cathode buffer: 0.025 M Tris/0.04 M aminocaproicacid/20% methanol). The five % skimmed milk, diluted with TBS-T (TBSbuffer, 0.1% Tween 20), was added to the transcribed PVDF membrane andsubjected to a reaction at room temperature for 1 hour for blocking, andthen 2 mL of the anti-human Lamp-1 antibody (produced by BDBiosciences), diluted 250-fold with PBS-T, or the 250-fold dilutedanti-human Flotillin-2 antibody (produced by BD Biosciences) weresubjected to a reaction at room temperature for 1 hour. The PVDFmembrane after the reaction was washed three times with TBS-T, andsubjected to a reaction with the secondary antibody {anti-mouse IgG(H+L), rabbit, IgG fraction, peroxidase-conjugated antibody} (producedby Wako Pure Chemical Industries Ltd.), diluted 5,000-fold with PBS-T,at room temperature for 1 hour. After washing five times with TBS-T, theImunoStar Zeta (produced by Wako Pure Chemical Industries, Ltd.) wasadded to the PVDF membrane, after subjected to a reaction with eachantibody, and a luminescent signal was detected using the LAS-4000(manufactured by GE Healthcare).

It should be noted that the types of Tim proteins, the carrier and theeluents used for obtaining the extracellular membrane vesicles from theTim protein-bound (non-bound) carrier, used in each Example andComparative Example, are shown in the following Table 23.

TABLE 23 Comparative Example Example 14 15 56 57 58 59 60 61 62 63 64 6566 67 Tim protein- Carrier Dynabeads Protein G beads (non-)bound Fc tagfusion type — Human- Mouse- Human- Mouse- Human- Mouse- carrier Timprotein derived derived derived derived derived derived Tim-1 Tim-1Tim-3 Tim-3 Tim-4 Tim-4 Eluent 1% 1 mM 1% 1 mM 1% 1 mM 1% 1 mM 1% 1 mM1% 1 mM 1% 1 mM SDS EDTA SDS EDTA SDS EDTA SDS EDTA SDS EDTA SDS EDTASDS EDTA Lane numbers in FIG. 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14<Results>

The results of Western blotting obtained are shown in FIG. 14. In FIG.14, each lane is as follows.

Lane 1: Results of Comparative Example 14 (the results of the case wherethe carrier of only the magnetic beads of the Dynabeads protein G wasused, and the 1% SDS aqueous solution was used as the eluent);

Lane 2: Results of Comparative Example 15 (the results of the case wherethe carrier of only the magnetic beads of the Dynabeads protein G wasused and the TBS solution containing 1 mM EDTA was used as the eluent);

Lane 3: Results of Example 56 (the results of the case of using thecarrier in which the human-derived Tim-1 protein was bound to theDynabeads protein G, and using the 1% SDS aqueous solution as theeluent);

Lane 4: Results of Example 57 (the results of the case of using thecarrier in which the human-derived Tim-1 protein was bound to theDynabeads protein G, and using the TBS solution containing 1 mM EDTA asthe eluent);

Lane 5: Results of Example 58 (the results of the case of using thecarrier in which the mouse-derived Tim-1 protein was bound to theDynabeads protein G, and using the 1% SDS aqueous solution as theeluent);

Lane 6: Result of Example 59 (the results in a case of using the carrierin which the mouse-derived Tim-1 protein was bound to the Dynabeadsprotein G, and using the TBS solution containing 1 mM EDTA as theeluent);

Lane 7: Results of Example 60 (the results of the case of using thecarrier on which the human-derived Tim-3 protein was bound to theDynabeads protein G, and using the 1% SDS aqueous solution as theeluent);

Lane 8: Results of Example 61 (the results of the case of using thecarrier in which the human-derived Tim-3 protein was bound to theDynabeads protein G, and using the TBS solution containing 1 mM EDTA asthe eluent);

Lane 9: Results of Example 62 (the results of the case of using thecarrier in which the mouse-derived Tim-3 protein was bound to theDynabeads protein G, and using the 1% SDS aqueous solution as theeluent); Lane 10: Results of Example 63 (the results of the case ofusing the carrier in which the mouse-derived Tim-3 protein was bound tothe Dynabeads protein G, and using the TBS solution containing 1 mM EDTAas the eluent);

Lane 11: Results of Example 64 (the results of the case of using thecarrier in which the human-derived Tim-4 protein was bound to theDynabeads protein G, and using the 1% SDS aqueous solution as theeluent);

Lane 12: Results of Example 65 (the results of the case of using thecarrier in which the human-derived Tim-4 protein was bound to theDynabeads protein G, and using the TBS solution containing 1 mM EDTA asthe eluent);

Lane 13: Results of Example 66 (the results of the case of using thecarrier in which the mouse-derived Tim-4 protein was bound to theDynabeads protein G, and using the 1% SDS aqueous solution as theeluent);

Lane 14: Results of Example 67 (the results in a case of using thecarrier in which the mouse-derived Tim-4 protein was bound to theDynabeads protein G, and using the TBS solution containing 1 mM EDTA asthe eluent).

From FIG. 14, it has been revealed the extracellular membrane vesiclescan be obtained by using the carrier on which the Tim-1 protein, theTim-3 protein or the Tim-4 protein is bound (the Tim protein-boundcarrier), regardless of the origin of the protein or the elution method,from the fact that a band of Lamp-1 or Flotillin-2, which is exosomemarker protein, was observed in lanes 3 to 14.

In addition, it has been revealed that, in the case of eluting with SDS,more extracellular membrane vesicles can be obtained in the order of theTim-4 protein-bound carrier>the Tim-1 protein-bound carrier>the Tim-3protein-bound carrier.

Examples 68 to 79. Obtaining of Extracellular Membrane Vesicles byCarrier on which Tim Protein is Immobilized

Obtaining of the extracellular membrane vesicles pertaining to thepresent invention was carried out, using the carriers on which each ofthe biotin-labeled Tim-1 protein, the biotin-labeled Tim-3 protein orthe biotin-labeled Tim-4 protein was immobilized.

<(1) Preparation of Culture Supernatant Sample>

The calcium ion-containing culture supernatant (stock solution) wasobtained by the same method as in “(1) preparation of calciumion-containing culture supernatant (stock solution)” in Examples 50 to67, and Comparative Examples 14 to 15.

<(2) Biotin Labeling of SH-Group of Fc Tag Fusion Type Tim-1 Protein>

For 200 μL of the PBS solution containing the Fc tag fusion typemouse-derived Tim-1 protein (produced by Wako Pure Chemical Industries,Ltd.) (containing 20 μg of the Fc tag fusion type mouse-derived Tim-1protein), the SH-group of the Fc tag fusion type mouse-derived Tim-1protein was labeled with biotin using the Biotin Labeling Kit-SH(produced by Dojindo Molecular Technologies, Inc.), according to aprotocol attached to the kit to obtain 200 μL of the PBS solutioncontaining the SH-group biotin-labeled Fc tag fusion type mouse-derivedTim-1 protein.

<(3) Biotin Labeling of SH-Group of Fc Tag Fusion Type Tim-3 Protein>

For 200 μL of the PBS solution containing the Fc tag fusion typemouse-derived Tim-3 protein (produced by Wako Pure Chemical Industries,Ltd.) (containing 20 μg of the Fc tag fusion type mouse-derived Tim-3protein), the SH-group of the Fc tag fusion type mouse-derived Tim-3protein was labeled with biotin using the Biotin Labeling Kit-SH(produced by Dojindo Molecular Technologies, Inc.), according to aprotocol attached to the kit to obtain 200 μL of the PBS solutioncontaining the SH-group biotin-labeled Fc tag fusion type mouse-derivedTim-3 protein.

<(4) Biotin Labeling of SH-Group of Fc Tag Fusion Type Tim-4 Protein>

For 100 μL of the PBS solution containing the Fc tag fusion typemouse-derived Tim-4 protein (produced by Wako Pure Chemical Industries,Ltd.) (containing 10 μg of the Fc tag fusion type mouse-derived Tim-4protein), the SH-group of the Fc tag fusion type mouse-derived Tim-4protein was labeled with biotin using the Biotin Labeling Kit-SH(produced by Dojindo Molecular Technologies, Inc.), according to aprotocol attached to the kit to obtain 100 μL of the PBS solutioncontaining the SH-group biotin-labeled Fc tag fusion type mouse-derivedTim-4 protein.

<(5) Dilution of Tag-Fused Tim Protein>

The PBS solution (10 μL) containing the Fc tag fusion type mTim-1protein (produced by Wako Pure Chemical Industries, Ltd.) (containing 1μg of the Fc tag fusion type mTim-1 protein) was mixed with 190 μL ofTBS to obtain 200 μL of a solution containing 1 μg of thebiotin-unlabeled Fc tag fusion type mTim-1 protein.

The TBS solution (11.5 μL) containing the SH-group biotin-labeled Fc tagfusion type mTim-1 protein prepared in the (2) (containing 1 μg of theSH-group biotin-labeled Fc tag fusion type mTim-1 protein) was mixedwith 188.5 μL of TBS to obtain 200 μL of a solution containing 1 μg ofthe biotin-labeled Fc tag fusion type mTim-1 protein.

The PBS solution (10 μL) containing the Fc tag fusion type mTim-3protein (produced by Wako Pure Chemical Industries, Ltd.) (containing 1μg of the Fc tag fusion type mTim-3 protein) was mixed with 190 μL ofTBS to obtain 200 μL of a solution containing 1 μg of thebiotin-unlabeled Fc tag fusion type mTim-3 protein.

The TBS solution (9.0 μL) containing the SH-group biotin-labeled Fc tagfusion type mTim-3 protein prepared in the (3) (containing 1 μg of theSH-group biotin-labeled Fc tag fusion type mTim-3 protein) was mixedwith 191.0 μL of TBS to obtain 200 μL of a solution containing 1 μg ofthe biotin-labeled Fc tag fusion type mTim-3 protein.

The PBS solution (10 μL) containing the Fc tag fusion type mTim-4protein (produced by Wako Pure Chemical Industries, Ltd.) (containing 1μg of the Fc tag fusion type mTim-4 protein) was mixed with 190 μL ofTBS to obtain 200 μL of a solution containing 1 μg of thebiotin-unlabeled Fc tag fusion type mTim-4 protein.

The TBS solution (10 μL) containing the SH-group biotin-labeled Fc tagfusion type mTim-4 protein prepared in the (4) (containing 1 μg of theSH-group biotin-labeled Fc tag fusion type mTim-4 protein) was mixedwith 190 μL of TBS to obtain 200 μL of a solution containing 1 μg of thebiotin-labeled Fc tag fusion type mTim-4 protein.

<(6) Washing of Beads>

The PBS-T solution (20 μL) containing 30 μg/μL of the Dynabeads ProteinG (produced by Thermo Fisher Scientific Inc.) (containing 0.6 mg of theDynabeads Protein G) was transferred into six 1.5 mL tubes (manufacturedby BM Equipment Co., Ltd.), and each subjected to the washing operation.

In addition, 60 μL of PBS solution containing the 10 μg/μL DynabeadsM-270 Streptavidin C1 (produced by Thermo Fisher Scientific Inc.)(containing 0.6 mg of the Dynabeads M-270 Streptavidin C1) was dispensedinto each of the 1.5 mL tubes (manufactured by BM Equipment Co., Ltd.),and the washing operation was carried out by the same method as aboveusing 500 μL of TBS.

<(7) Immobilization of Fc Tag Fusion Type Tim Protein on Beads>

The solution (200 μL) containing 1 μg of the SH-group biotin-unlabeledFc tag fusion type mTim-1 protein, 200 μL of the solution containing 1μg of the SH-group biotin-unlabeled Fc tag fusion type mTim-3 protein,or 200 μL of the solution containing 1 μg of the SH-groupbiotin-unlabeled Fc tag fusion type mTim-4 protein was each added to six1.5 mL tubes containing the 0.6 mg of pellet-like Dynabeads Protein G,after the washing operation, and subjected to a reaction at 8° C. for 1hour to obtain each of the carrier on which the SH-groupbiotin-unlabeled Fc tag fusion type mTim-1 protein was bound (it may beabbreviated as “the biotin-unlabeled Fc tag fusion type mTim-1 carrier”,in some cases), the carrier on which the SH-group biotin-unlabeled Fctag fusion type mTim-3 protein was bound (it may be abbreviated as “thebiotin-unlabeled Fc tag fusion type mTim-3 carrier”, in some cases), andthe carrier on which the SH-group biotin-unlabeled Fc tag fusion typemTim-4 protein was bound (it may be abbreviated as “the biotin-unlabeledFc tag fusion type mTim-4 carrier”, in some cases).

In addition, 200 μL of the solution containing 1 μg of the SH-groupbiotin-labeled Fc tag fusion type mTim-1 protein, 200 μL of a solutioncontaining 1 μg of the SH-group biotin-labeled Fc tag fusion type mTim-3protein, or 200 μL of a solution containing 1 μg of the SH-groupbiotin-labeled Fc tag fusion type mTim-4 protein was added to the 1.5 mLtube containing 0.6 mg of the pellet-like Dynabeads M270 StreptavidinC1, after the washing operation, to bind on the beads by the same methodas above to prepare each of the carrier on which the SH-groupbiotin-labeled Fc tag fusion type mTim-1 protein was bound (it may beabbreviated as “the biotin-labeled Fc tag fusion type mTim-1 carrier”,in some cases), the carrier on which the SH-group biotin-labeled Fc tagfusion type mTim-3 protein was bound (it may be abbreviated as “thebiotin-labeled Fc tag fusion type mTim-3 carrier”, in some cases), andthe carrier on which the SH-group biotin-labeled Fc tag fusion typemTim-4 protein was bound (it may be abbreviated as “the biotin-labeledFc tag fusion type mTim-4 carrier”, in some cases).

It should be noted that, the resulting these six types of the carriersmay be abbreviated collectively as “the Tim protein-bound carrier” insome cases.

<(8) Obtaining of Extracellular Membrane Vesicles by Obtaining Method ofthe Present Invention>

The resulting these Tim protein-bound carriers above were subjected tothe washing operation three times with 500 μL of TBS-T (Tris buffer,0.1% Tween 20), and twice with 500 μL of TBS to obtain the Timprotein-bound carrier in a pellet state. Subsequently, 1 mL of thecalcium ion-containing culture supernatant (stock solution) was added toeach of the resulting six pellet-like Tim protein-bound carriers, andeach subjected to a reaction at 8° C. for 3 hours. After that, the Timprotein-bound carriers after the reaction were subjected to the washingoperation 3 times with each 500 μL of the calcium ion-containing TBS-T(Tris buffer, 0.0005% Tween 20, 2 mM CaCl₂). At the third time of thewashing operation, 500 μL of the CaCl₂-containing TBS-T solutioncontaining the Tim protein-bound carrier was dispensed into each of thetwo 1.5 mL tubes in an amount of each 250 μL, and the tubes were loadedon a magnetic stand, and then the washing solution was removed.

50 μL of 1% SDS aqueous solution or 25 μL of the TBS solution containing1 mM EDTA, as the eluent, was added to each 0.3 mg of the 6 types of theTim protein-bound carriers in a pellet state, and they were mixed usinga vortex mixer at room temperature for 10 seconds, and spun down. To thetube to which EDTA had been added, 25 μL of the TBS solution containing1 mM EDTA was added again, and the same operation was carried out toobtain the eluate.

It should be noted that the types of Tim proteins, the carriers, and theTim protein-(un) bound carriers, used in each Example, are shown in thefollowing Table 24.

TABLE 24 Example 68 69 70 71 72 73 74 75 76 77 78 79 Tim CarrierDynabeads Streptavidin Dynabeads Streptavidin Dynabeads Streptavidinprotein- Protein G beads beads Protein G beads beads Protein G beadsbeads bound Fc tag Biotin- SH-group Biotin- SH-group Biotin- SH-groupcarrier fusion type unlabeled biotin-labeled unlabeled biotin-labeledunlabeled biotin-labeled Tim protein mouse-derived mouse-derivedmouse-derived mouse-derived mouse-derived mouse-derived Tim-1 Tim-1Tim-3 Tim-3 Tim-4 Tim-4<(9) Western Blotting>

Western blotting was carried out by the same method as in “(5) Westernblotting” in Examples 56 to 67, except for using 11.25 μL of theresulting each eluate in the (6), instead of “11.25 μL of the resultingeach eluate in (4)”, in <(5) Western blotting> of Examples 56 to 67, andusing, as the secondary antibody, {anti-mouse IgG (H+L), donkey, IgGfraction, peroxidase-conjugated antibody}(produced by Jackson ImmunoResearch Inc.), diluted 30,000-fold with TBS-T, instead of “thesecondary antibody {anti-mouse IgG (H+L), rabbit, IgG fraction,peroxidase-conjugated antibody}(produced by Wako Pure ChemicalIndustries, Ltd.).

It should be noted that the types of Tim proteins, carriers, and theeluents used for obtaining the extracellular membrane vesicles from theTim protein-bound (non-bound) carrier, used in each Example, and thelane numbers in Western blotting are shown in the following Table 25.

TABLE 25 Example 68 69 70 71 72 73 74 75 76 77 78 79 Tim CarrierDynabeads Streptavidin Dynabeads Streptavidin Dynabeads Streptavidinprotein- Protein G beads beads Protein G beads beads Protein G beadsbeads bound Fc tag Biotin- SH-group Biotin- SH-group Biotin- SH-groupcarrier fusion type unlabeled biotin-labeled unlabeled biotin-labeledunlabeled biotin-labeled Tim protein mouse-derived mouse-derivedmouse-derived mouse-derived mouse-derived mouse-derived Tim-1 Tim-1Tim-3 Tim-3 Tim-4 Tim-4 Eluent 1% 1 mM 1% 1 mM 1% 1 mM 1% 1 mM 1% 1 mM1% 1 mM SDS EDTA SDS EDTA SDS EDTA SDS EDTA SDS EDTA SDS EDTA Lanenumbers in FIG. 15 1 2 3 4 5 6 7 8 9 10 11 12<Results>

The results of Western blotting obtained are shown in FIG. 15. In FIG.15, each lane is the result of the following.

Lane 1: Results of Example 68 (the results of the case of using thecarrier in which the SH-group biotin-unlabeled Fc tag fusion type mTim-1protein was bound on the Dynabeads protein G, and using the 1% SDSaqueous solution as the eluent);

Lane 2: Results of Example 69 (the results of the case of using acarrier in which the SH-group biotin-unlabeled Fc tag fusion type mTim-1protein was bound on the Dynabeads protein G, and using the TBS solutioncontaining 1 mM EDTA as the eluent);

Lane 3: Results of Example 70 (the results of the case of using acarrier in which the SH-group biotin-labeled Fc tag fusion type mTim-1protein was bound on the Dynabeads M270 Streptavidin, and using the 1%SDS aqueous solution as the eluent);

Lane 4: Results of Example 71 (the results of the case of using acarrier in which the SH-group biotin-labeled Fc tag fusion type mTim-1protein was bound on the Dynabeads M270 Streptavidin, and using the TBSsolution containing 1 mM EDTA as the eluent);

Lane 5: Results of Example 72 (the results of the case of using acarrier in which the SH-group biotin-unlabeled Fc tag fusion type mTim-3protein was bound on the Dynabeads protein G, and using the 1% SDSaqueous solution as the eluent);

Lane 6: Results of Example 73 (the results of the case of using acarrier in which the SH-group biotin-unlabeled Fc tag fusion type mTim-3protein was bound on the Dynabeads protein G, and using the TBS solutioncontaining 1 mM EDTA as the eluent);

Lane 7: Results of Example 74 (the results of the case of using acarrier in which the SH-group biotin-labeled Fc tag fusion type mTim-3protein was bound on the Dynabeads M270 Streptavidin, and using the 1%SDS aqueous solution as the eluent);

Lane 8: Results of Example 75 (the results of the case of using acarrier in which the SH-group biotin-labeled Fc tag fusion type mTim-3protein was bound on the Dynabeads M270 Streptavidin, and using the TBSsolution containing 1 mM EDTA as the eluent);

Lane 9: Results of Example 76 (the results of the case of using acarrier in which the SH-group biotin-unlabeled Fc tag fusion type mTim-4protein was bound on the Dynabeads protein G, and using the 1% SDSaqueous solution as the eluent);

Lane 10: Results of Example 77 (the results of the case of using acarrier in which the SH-group biotin-unlabeled Fc tag fusion type mTim-4protein was bound on the Dynabeads protein G, and using the TBS solutioncontaining 1 mM EDTA as the eluent);

Lane 11: Results of Example 78 (the results of the case of using acarrier in which the SH-group biotin-labeled Fc tag fusion type mTim-4protein was bound on the Dynabeads M270 Streptavidin, and using the 1%SDS aqueous solution as the eluent);

Lane 12: Results of Example 79 (the results of the case of using acarrier in which the SH-group biotin-labeled Fc tag fusion type mTim-4protein was bound on the Dynabeads M270 Streptavidin, and using the TBSsolution containing 1 mM EDTA as the eluent).

From FIG. 15, it has been revealed that the extracellular membranevesicles can be obtained, by using the carrier on which the Tim-1, theTim-3, or the Tim-4 was bound (the Tim protein-bound carrier), from thefact that a band of Lamp-1, which is a marker protein of exosome, wasobserved in any of the lanes 1 to 12.

In addition, it has been revealed that more extracellular membranevesicles can be obtained in the order of the Tim-4 protein-boundcarrier>the Tim-1 protein-bound carrier>the Tim-3 protein-bound carrier.It has been revealed that when eluting with the calcium chelating agent,more extracellular membrane vesicles can be obtained by immobilizationon the beads (carriers) via the SH group of the Tim4 protein, ascompared with immobilizing via the tag (lanes 9 to 12).

Examples 80 to 83, Comparative Examples 16 to 19. Obtaining ofExtracellular Membrane Vesicles by Carrier on which Tim Family Proteinwas Immobilized

Obtaining of the extracellular membrane vesicles pertaining to thepresent invention was carried out using the carrier in which the Tim-1protein, the Tim-2 protein, or the Tim-4 protein was immobilized, as theTim family protein, on the beads.

<(1) Preparation of Calcium Ion-Containing Culture Supernatant Sample>

The calcium ion-containing culture supernatant sample was obtained bythe same method as in “(1) Preparation of culture supernatant sample” ofExamples 1 to 8.

<(2) Washing of Beads>

The PBS-T solution (20 μL) containing the 30 μg/μL Dynabeads Protein G(produced by Thermo Fisher Scientific Inc.) (containing 4.8 mg of theDynabeads Protein G) was dispensed into each of the four 1.5 mL tubes(manufactured by BM Equipment Co., Ltd.), and each was subjected to thewashing operation.

<(3) Immobilization of 6×His Tag Fusion Type Tim Family Protein onBeads>

The TBS solution (100 μL) containing 4 μg of the anti-6×His antibody(clone No. 28 to 75, produced by Wako Pure Chemical Industries, Ltd.)was added to each of the four 1.5 mL tubes containing 0.6 mg of theDynabeads Protein G, in a pellet state, after the washing operation, andsubjected to a reaction at 8° C. for 30 minutes to obtain 100 μL of theTBS solution containing the carrier on which the anti-6×His antibody wasbound. These carriers on which the anti-6×His antibody was bound weresubjected to the washing operation three times with each 250 μL of TBS-T(Tris buffer, 0.1% Tween 20), and then subjected to the washingoperation twice with each 250 μL of TBS.

Next, 100 μL of the TBS solution containing 1 μg of the 6×His tag fusiontype mouse-derived Tim-1 protein (produced by R & D Systems Inc.), 100μL of the TBS solution containing 1 μg of the 6×His tag fusion typemouse-derived Tim-2 protein (produced by R & D Systems Inc.), or 100 μLof the TBS solution containing 1 μg of the 6×His tag fusion typemouse-derived Tim-4 protein (produced by R & D Systems Inc.) was eachadded to the anti-6×His antibody-bound Dynabeads Protein G carrier afterthe washing operation and subjected to a reaction at 8° C. for 1 hour toobtain each of the carrier on which the Tim-1 protein was bound via theanti-6×His antibody (it may be abbreviated as “the 6×His tag fusion typemTim-1 protein-bound carrier”, in some cases), the carrier on which theTim-2 protein was bound (it may be abbreviated as “the 6×His tag fusiontype mTim-2 protein-bound carrier”), and the carrier on which the Tim-4protein was bound (it may be abbreviated as “the 6×His tag fusion typemTim-4 protein-bound carrier”, in some cases).

It should be noted that the resulting 6×His fusion type mTim-1protein-bound carrier, and the 6×His fusion type mTim-4 protein-boundcarrier may be abbreviated collectively as “the Tim protein-boundcarrier” in some cases.

The types of the Tim proteins, the carrier and the Tim familyprotein-(non-)bound carriers, used in each Example, are shown in thefollowing Table 26.

TABLE 26 Comparative Comparative Example Example Comparative ComparativeExample Example Example 16 Example 17 80 81 Example 18 Example 19 82 83Tim family Carrier Anti-6xHis tag antibody-bound Dynabeads Protein Gcarrier protein- Tim family — — 6xHis tag fusion type 6xHis tag fusionType 6xHis tag fusion type (non-)bound protein mouse-derivedmouse-derived mouse-derived Tm-4 carrier Tm-1 protein Tm-2 proteinprotein<(4) Obtaining of Extracellular Membrane Vesicles by Obtaining Method ofthe Present Invention>

The resulting anti-6×His antibody-bound Dynabeads Protein G carrier, the6×His tag fusion type mTim-1 protein-bound carrier, the 6×His tag fusiontype mTim-2 protein-bound carrier, the 6×His tag fusion type mTim-2protein-bound carrier in the (3) were each subjected to the washingoperation three times with 250 μL of TBS-T (Tris buffer, 0.1% Tween 20)and twice with 250 μL of TBS to obtain each carrier in a pellet state.Thereafter, 200 μL of the calcium ion-containing culture supernatantsample was added to each carrier in a pellet state, and subjected to areaction at 8° C. for 3 hours. Each carrier after the reaction wassubjected to the washing operation three times with 500 μL of thecalcium ion-containing TBS-T (Tris buffer, 0.0005% Tween 20, 2 mMCaCl₂). At the third time of the washing operation, 500 μL of theCaCl₂-containing TBS-T solution containing each carrier was dispensedinto two 1.5 mL tubes in an amount of each 250 μL, and after loading thetube on a magnetic stand, the washing solution was removed.

50 μL of the 1% SDS aqueous solution or 25 μL of the TBS solutioncontaining 1 mM EDTA, as the eluent, was added to each 0.3 mg of thecarrier in a pellet state, and they were mixed using a vortex mixer atroom temperature for 10 seconds, and spun down. To the tube to whichEDTA had been added, 25 μL of the TBS solution containing 1 mM EDTA wasadded again, and the same operation was carried out to obtain theeluate.

<(5) Western Blotting>

Western blotting was carried out by the same method as in “(7) Westernblotting” in Examples 68 to 79, except for using 11.25 μL of theresulting each eluate in the (4).

It should be noted that the types of the Tim family proteins, thecarrier, and the eluents used for obtaining the extracellular membranevesicles from the Tim protein-bound (non-bound) carrier, used in eachExample, and the lane numbers in Western blotting are shown in thefollowing Table 27.

TABLE 27 Comparative Comparative Example Example Comparative ComparativeExample Example Example 16 Example 17 80 81 Example 18 Example 19 82 83Tim family Carrier Anti-6xHis tag antibody-bound Dynabeads Protein Gcarrier protein- Tim family — — 6xHis tag fusion type 6xHis tag fusionType 6xHis tag fusion type (non-)bound protein mouse-derivedmouse-derived mouse-derived Tm-4 carrier Tm-1 protein Tm-2 proteinprotein Eluent 1% 1 mM 1% 1 mM 1% 1 mM 1% 1 mM SDS EDTA SDS EDTA SDSEDTA SDS EDTA Lane numbers in FIG. 16 1 2 3 4 5 6 7 8<Results>

The results of Western blotting obtained are shown in FIG. 16. In FIG.16, each lane shows the following result.

Lane 1: Results of Comparative Example 16 (the results of the case ofusing the carrier in which the anti-6×His antibody was bound on theDynabeads protein G, and using the 1% SDS aqueous solution as theeluent);

Lane 2: Results of Comparative Example 17 (the results of the case ofusing the carrier in which the anti-6×His antibody was bound to theDynabeads protein G, and using the TBS solution containing 1 mM EDTA asthe eluent);

Lane 3: Results of Example 80 (the results of the case of using thecarrier in which the 6×His tag fusion type mTim-1 protein was bound onthe Dynabeads protein G via the anti-6×His antibody, and using the 1%SDS aqueous solution as the eluent);

Lane 4: Results of Example 81 (the results of the case of using thecarrier in which the 6×His tag fusion type mTim-1 protein was bound onthe Dynabeads protein G via the anti-6×His antibody, and using the TBSsolution containing 1 mM EDTA as the eluent);

Lane 5: Results of Comparative Example 18 (the results of the case ofusing the carrier in which the 6×His tag fusion type mTim-2 protein wasbound on the Dynabeads protein G via the anti-6×His antibody, and usingthe 1% SDS aqueous solution as the eluent);

Lane 6: Results of Comparative Example 19 (the results of the case ofusing the carrier in which the 6×His tag fusion type mTim-2 protein wasbound on the Dynabeads protein G via the anti-6×His antibody, and usingthe TBS solution containing 1 mM EDTA as the eluent);

Lane 7: Results of Example 82 (the results of the case of using thecarrier in which the 6×His tag fusion type mTim-4 protein was bound onthe Dynabeads protein G via the anti-6×His antibody, and using the 1%SDS aqueous solution as the eluent);

Lane 8: Result of Example 83 (the results of the case of using thecarrier in which the 6×His tag fusion type mTim-4 protein was bound onthe Dynabeads protein G via the anti-6×His antibody, and using the TBSsolution containing 1 mM EDTA as the eluent).

From FIG. 16, in the case of binding the 6×His tag fusion type Tim-2protein, a band of Lamp-1, which is a marker protein of exosome, was notobserved in any of the elution methods (lanes 5 to 6). On the otherhand, in the case of the carrier on which the 6×His tag fusion typeTim-1 protein or the 6×His tag fusion type Tim-4 protein was bound, theband of anti-Lamp-1, which is a marker protein of exosome, was observedin any of the elution methods (lanes 3 to 4, lanes 7 to 8). From theseresults, it has been revealed that the extracellular membrane vesiclescannot be obtained, using the carrier on which the Tim-2 protein wasimmobilized.

In addition, it has been revealed that, when eluting by SDS, moreextracellular membrane vesicles can be obtained in the order of thecarrier on which the Tim-4 protein is bound>carrier on which the Tim-1protein is bound.

Examples 84 to 95, Comparative Examples 20 to 21. Obtaining of Virusesby Carrier on which Tim Protein is Immobilized

As described below, obtaining of the viruses pertaining to the presentinvention was carried out using the carrier on which the Tim-1 protein,the Tim-3 protein, or the Tim-4 protein, as Tim protein, was eachimmobilized on the beads.

<(1) Preparation of Co-Transfection Cell Culture Supernatant>

A PSFM-J1 medium (produced by Wako Pure Chemical Industries, Ltd.)containing about 2 μg of mouse IL23 expression vector DNA, 90 ng ofLinear AcNPV DNA, and 3 μL of TransiT-Insect (produced by Mirus Bio LLC)was added to 1.0×10⁶ cells of Sf9 cell seeded in a 25 cm² flask. Afterculturing for 7 days at 28° C., the culture supernatant (co-transfectionsolution) was recovered.

<(2) Preparation of Baculovirus-Containing Insect Cell CultureSupernatant Sample>

Sf9 cells diluted with the PSFM-J 1 medium, so as to attain 1.5×10⁶cells/mL (50 mL), were prepared. The co-transfection solution of 1/200volume of the culture solution was added thereto, and cultured at 27° C.under shaking at 130 rpm. After 72 hours, the cell culture solution wasrecovered, and subjected to centrifugal separation at 3,000×G, for 30minutes at 4° C., to fractionate into precipitate and supernatant. Theresulting supernatant was used as a recombinant baculovirus solutionexpressing mouse IL-23 (hereinafter it may be abbreviated as “thebaculovirus solution”, in some cases).

In addition, CaCl₂ was added to the resulting baculovirus solution, soas to attain the final concentration of 2 mM to obtain the baculovirussolution containing 2 mM CaCl₂ (hereinafter it may be abbreviated as“the calcium ion-containing baculovirus solution”, in some cases).

<(3) Washing of Beads>

Washing of the beads was carried out by the same method as in “(2)Washing of beads” of Examples 56 to 67 and Comparative Examples 14 to15.

<(4) Immobilization of Fc Tag Fusion Type Tim Protein on Beads>

As shown in the following Table 28, 6 types of the Tim protein-boundcarriers were obtained by the same method as in “(3) Immobilization ofFc tag fusion type Tim protein on beads” of Examples 56 to 67 andComparative Examples 14 to 15.

TABLE 28 1 2 3 4 5 6 Tim protein- Carrier Dynabeads Protein G beadsbound carrier Fc tag fusion type Human- Mouse- Human- Mouse- Human-Mouse- Tim protein derived derived derived derived derived derived Tim-1Tim-1 Tim-3 Tim-3 Tim-4 Tim-4<(5) Obtaining of Baculovirus by Method of the Present Invention>

Each eluate was obtained by carrying out similarly as in “(4) Obtainingof extracellular membrane vesicles by method of the present invention”in Examples 56 to 67 and Comparative Examples 14 to 15, except for using“1 mL of the calcium ion-containing baculovirus solution prepared in the(2)”, instead of “1 mL of the calcium ion-containing culturesupernatant”.

<(6) Western Blotting>

Western blotting was carried out by the same method as in “(5) Westernblotting” of Examples 56 to 67 and Comparative Examples 14 to 15, exceptfor using “the anti-baculovirus gp64 antibody (produced by NovusBiologicals LLC), diluted 500-fold with TBS-T”, instead of “2 mL of theanti-human Lamp-1 antibody, or diluted 250-fold with TBS-T (produced byBD Biosciences), and 2 mL of the 250-fold diluted anti-human Flotillin-2antibody (produced by BD Biosciences)”.

It should be noted that the types of Tim proteins, carrier, and theeluents used for obtaining the extracellular membrane vesicles from theTim protein-bound (non-bound) carrier, used in each Example andComparative Example, and the lane numbers in Western blotting are shownin the following Table 29.

TABLE 29 Comparative Example Example 20 21 84 85 86 87 88 89 90 91 92 9394 95 Tim protein- Carrier Dynabeads Protein G beads (non-)bound Fc tag— Human- Mouse- Human- Mouse- Human- Mouse- carrier fusion type Derivedderived Derived derived Derived derived Tim protein Tim-1 Tim-1 Tim-3Tim-3 Tim-4 Tim-4 (hTim-1) (mTim-1) (hTim-3) (mTim-3) (hTim-4) (mTim-4)Eluent 1% 1 mM 1% 1 mM 1% 1 mM 1% 1 mM 1% 1 mM 1% 1 mM 1% 1 mM SDS EDTASDS EDTA SDS EDTA SDS EDTA SDS EDTA SDS EDTA SDS EDTA Lane numbers inFIG. 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14<Results>

The results of Western blotting obtained are shown in FIG. 17. In FIG.17, each lane shows the following result.

Lane 1: Results of Comparative Example 20 (the results of the case whereonly the magnetic beads carrier of the Dynabeads protein G was used, andthe 1% SDS aqueous solution was used as the eluent);

Lane 2: Results of Comparative Example 21 (the results of the case whereonly the magnetic beads carrier of the Dynabeads protein G was used, andthe TBS solution containing 1 mM EDTA was used as the eluent);

Lane 3: Results of Example 84 (the results of the case of using thecarrier in which the hTim-1 protein was bound on the Dynabeads proteinG, and using the 1% SDS aqueous solution as the eluent);

Lane 4: Results of Example 85 (the results of the case of using thecarrier in which the hTim-1 protein was bound on the Dynabeads proteinG, and using the TBS solution containing 1 mM EDTA as the eluent);

Lane 5: Results of Example 86 (the results of the case of using thecarrier in which the mTim-1 protein was bound on the Dynabeads proteinG, and using the 1% SDS aqueous solution as the eluent);

Lane 6: Results of Example 87 (the results of the case of using thecarrier in which the mTim-1 protein was bound on the Dynabeads proteinG, and using the TBS solution containing 1 mM EDTA as the eluent);

Lane 7: Results of Example 88 (the results of the case of using thecarrier in which the hTim-3 protein was bound on the Dynabeads proteinG, and using the 1% SDS aqueous solution as the eluent);

Lane 8: Results of Example 89 (the results of the case of using thecarrier in which the hTim-3 protein was bound on the Dynabeads proteinG, and using the TBS solution containing 1 mM EDTA as the eluent);

Lane 9: Results of Example 90 (the results of the case of using thecarrier in which the mTim-3 protein was bound on the Dynabeads proteinG, and using the 1% SDS aqueous solution as the eluent);

Lane 10: Results of Example 91 (the results of the case of using thecarrier in which the mTim-3 protein was bound on the Dynabeads proteinG, and using the TBS solution containing 1 mM EDTA as the eluent);

Lane 11: Results of Example 92 (the results of the case of using thecarrier in which the hTim-4 protein was bound on the Dynabeads proteinG, and using the 1% SDS aqueous solution as the eluent);

Lane 12: Results of Example 93 (the results of the case of using thecarrier in which the hTim-4 protein was bound to the Dynabeads proteinG, and using the TBS solution containing 1 mM EDTA as the eluent);

Lane 13: Results of Example 94 (the results of the case of using thecarrier in which the mTim-4 protein was bound on the Dynabeads proteinG, and using the 1% SDS aqueous solution as the eluent);

Lane 14: Results of Example 95 (the results of the case of using thecarrier in which the mTim-4 protein was bound on the Dynabeads proteinG, and using the TBS solution containing 1 mM EDTA as the eluent).

From FIG. 17, it has been revealed that the baculovirus can be obtainedby using the carrier on which the Tim-1 protein, the Tim-3 protein orthe Tim-4 protein is bound (the Tim protein-bound carrier), regardlessof the origin of the Tim protein and the elution method, from the factthat a band of baculovirus marker protein gp64 was observed in the lanes3 to 14.

In addition, it has been revealed that, when eluting by SDS, moreviruses can be obtained in the order of the carrier on which the Tim3protein is bound and the carrier on which the Tim-4 protein is bound inthe same level, next the carrier on which the Tim-1 protein is bound.

It has been revealed that, when eluting using the calcium ion chelatingagent, more viruses can be obtained in the order of the carrier on whichthe Tim-3 protein is bound, followed by the carrier on which the Tim-1protein is bound, and the carrier to which the Tim-4 protein is bound.

Examples 96 to 99, Comparative Examples 22-25. Obtaining of Viruses byCarrier on which Tim Family Protein is Immobilized

As described below, obtaining of the viruses pertaining to the presentinvention was carried out using the carrier in which each of the Tim-1protein, the Tim-2 protein, or the Tim-4 protein, as the Tim familyprotein, was immobilized on the beads.

<(1) Preparation of Co-Transfection Cell Culture Supernatant>

Preparation of the co-transfected cell culture supernatant was carriedout similarly as in “(1) Preparation of co-transfection cell culturesupernatant” of Examples 84 to 95 and Comparative Examples 20 to 21.

<(2) Preparation of Calcium Ion-Containing and Baculovirus-ContainingInsect Cell Culture Supernatant Sample>

The calcium ion-containing baculovirus solution was obtained by the samemethod as in “(2) Preparation of baculovirus-containing insect cellculture supernatant sample” of Examples 84 to 95 and ComparativeExamples 20 to 21.

<(3) Washing of Beads>

The PBS-T solution (20 μL) containing the 30 μg/μL Dynabeads Protein G(produced by Thermo Fisher Scientific Inc.) (containing 4.8 mg of theDynabeads Protein G) was dispensed into four 1.5 mL tubes (produced byBM Equipment Co., Ltd.), and each was subjected to the washingoperation.

<(4) Immobilization of 6×His Tag Fusion Type Tim Family Protein onBeads>

The carrier on which the 6×His tag fusion type mTim-1 protein is bound,the carrier on which the 6×His tag fusion type mTim-2 protein is bound,and the carrier on which the 6×His tag fusion type mTim-4 protein isbound were each obtained by carrying out similarly as in “(3)Immobilization of 6×His tag fusion type Tim family protein on beads” ofExamples 80 to 83 and Comparative Examples 16 to 19.

The types of the Tim family proteins, the carrier and the Tim familyprotein-(non-)bound carrier, used in each Example, are shown in thefollowing Table 30.

TABLE 30 Comparative Comparative Example Example Comparative ComparativeExample Example Example 22 Example 23 96 97 Example 24 Example 25 98 99Tim family Carrier Anti-6xHis tag antibody-bound Dynabeads Protein Gcarrier protein- Tim family — — 6xHis tag fusion type 6xHis tag fusionType 6xHis tag fusion type bound carrier protein mouse-derivedmouse-derived mouse-derived Tim-4 Tim-1 protein Tim-2 protein protein<(5) Obtaining of Baculovirus by Obtaining Method of the PresentInvention>

An eluate was obtained by carrying out the same method as in “(4)Obtaining of extracellular membrane vesicles by obtaining method of thepresent invention” of Examples 80 to 83 and Comparative Examples 16 to19, except for using “200 μL of the calcium ion-containing baculovirussolution” prepared in the (2), instead of “200 μL of the calciumion-containing cell culture supernatant sample”.

<(6) Western Blotting>

Western blotting was carried out by the same method as in “(5) Westernblotting” in Examples 80 to 83 and Comparative Examples 16 to 19, exceptfor “adding 1 μL of the 4× sample buffer (produced by Wako Pure ChemicalIndustries, Ltd.) to 3 μL of the resulting each eluate obtained by the(5) Obtaining of baculovirus by obtaining method of the presentinvention”, instead of “3.75 μL of the 4× sample buffer (produced byWako Pure Chemical Industries, Ltd.) to 11.25 μL of the resulting eacheluate”, using “4 μL of each sample for Western blotting”, instead of“15 μL of each sample for Western blotting”, and using “theanti-baculovirus gp64 antibody” instead of “the anti-Lamp-1 antibody”.

It should be noted that the types of Tim family proteins, carrier andthe eluents used to obtain the extracellular membrane vesicles from theTim family protein-(non-)bound carriers, used in each Example, and thelane numbers in Western blotting are shown in the following Table 31.

TABLE 31 Comparative Comparative Example Example Comparative ComparativeExample Example Example 22 Example 23 96 97 Example 24 Example 25 98 99Tim family Carrier Anti-6xHis tag antibody-bound Dynabeads Protein Gcarrier protein- Tim — — 6xHis tag fusion type 6xHis tag fusion Type6xHis tag fusion type bound carrier protein mouse-derived mouse-derivedmouse-derived Tim-4 Tim-1 protein Tim-2 protein protein Eluent 1% 1 mM1% 1 mM 1% 1 mM 1% 1 mM SDS EDTA SDS EDTA SDS EDTA SDS EDTA Lane numbersin FIG. 18 1 2 3 4 5 6 7 8<Results>

The results of Western blotting obtained are shown in FIG. 18. In FIG.18, each lane shows the following result.

Lane 1: Results of Comparative Example 22 (the results of the case ofusing the carrier in which the anti-6×His tag antibody was bound on theDynabeads protein G, and using the 1% SDS aqueous solution as theeluent);

Lane 2: Results of Comparative Example 23 (the results of the case ofusing the carrier in which the anti-6×His tag antibody was bound on theDynabeads protein G, and using the TBS solution containing 1 mM EDTA asthe eluent);

Lane 3: Results of Example 96 (the results of the case of using acarrier in which the 6×His tag fusion type mTim-1 protein was bound onthe Dynabeads protein G via the anti-6×His tag antibody, and using the1% SDS aqueous solution as the eluent);

Lane 4: Result of Example 97 (the results of the case of using thecarrier in which the 6×His tag fusion type mTim-1 protein was bound onthe Dynabeads protein G via the anti-6×His tag antibody, and using theTBS solution containing 1 mM EDTA as the eluent);

Lane 5: Result of Comparative Example 24 (the results of the case ofusing the carrier in which the 6×His tag fusion type mTim-2 protein wasbound on the Dynabeads protein G via the anti-6×His tag antibody, andusing the 1% SDS aqueous solution as the eluent);

Lane 6: the result of Comparative Example 25 (the results of the case ofusing the carrier in which the 6×His tag fusion type mTim-2 protein wasbound on the Dynabeads protein G via the anti-6×His tag antibody, andusing the TBS solution containing 1 mM EDTA as the eluent);

Lane 7: Results of Example 98 (the results of the case of using thecarrier in which the 6×His tag fusion type mTim-4 protein was bound onthe Dynabeads protein G via the anti-6×His tag antibody, and using the1% SDS aqueous solution as the eluent);

Lane 8: Results of Example 99 (the results of the case of using thecarrier in which the 6×His tag fusion type mTim-4 protein was bound onthe Dynabeads protein G via the anti-6×His tag antibody, and using theTBS solution containing 1 mM EDTA as the eluent).

From FIG. 18, when the Tim-2 protein was bound, no gp64 band ofbaculovirus marker protein was observed by any of the elution methods(lanes 5 to 6). On the other hand, the gp64 band was observed in any ofthe elution methods for the carrier on which the Tim-1 protein or theTim-4 protein was bound (lanes 3 to 4, lanes 7 to 8).

From this result, it has been revealed that it was impossible to obtainthe baculovirus with the carrier on which the Tim-2 protein wasimmobilized.

Examples 100 to 105, Comparative Examples 26 to 33. Comparison ofSensitivity in Detection of Extracellular Membrane Vesicles by ELISAUsing PS Protein

As described below, the extracellular membrane vesicles derived from theK562 cell culture supernatant was detected by carrying out sandwichELISA using a plate (well) on which an antibody against the PS proteinor an exosome surface marker protein (hereinafter it may be abbreviatedas “the marker protein”, in some cases) is immobilized, and using theHRP-labeled anti-CD63 antibody, as a detection antibody.

<(1) Preparation of Culture Supernatant (Stock Solution)>

The human chronic myelogenous leukemia cell strain K562, 1×10⁷ cells,secreting the extracellular membrane vesicles, was cultured using 80 mLof the X-VIVO 15 medium (produced by Lonza AG) for 3 days undercondition at 37° C. and 5% CO₂. Then the cells were precipitated bycentrifugal separation treatment (300×G, for 5 minutes) to remove thesupernatant. The precipitated cells were suspended in 60 mL of theX-VIVO 15 medium containing 10 μM monensin sodium (produced by MPBiomedicals Co., Ltd) and cultured for 24 hours under condition at 37°C. and 5% CO₂.

Thereafter, the culture solution was subjected to centrifugal separationtreatment (300×G, for 5 minutes) to recover the culture supernatant. Therecovered culture supernatant (60 mL) was further subjected tocentrifugal separation treatment three times (the first time: 300×G, for3 minutes, the second time: 1,200×G, for 20 minutes, the third time:10,000×G, for 20 minutes) to separate impurities and obtain asupernatant (hereinafter it may be abbreviated as “the culturesupernatant (stock solution)”, in some cases).

<(2) Preparation of Dilution Series of Ultracentrifugation PrecipitateFraction of Calcium Ion-Containing K562 Cell Culture Supernatant>

The resulting culture supernatant stock solution (10 mL) in the (1) wassubjected to centrifugal separation treatment (10,000×G, for 20 minutes)to separate impurities, and the supernatant was transferred to a newtube to obtain 10 mL of a centrifugal separation treated K562 cellculture supernatant. Next, 10 mL of the resulting centrifugal separationtreated K562 cell culture supernatant was subjected to ultracentrifugalseparation treatment (110,000×G, for 70 minutes), the supernatant wasremoved, and then 10 mL of TBS was added to the precipitate andsuspended. The suspension was subjected to ultracentrifugal separationtreatment (110,000×G, for 70 minutes), supernatant was removed, and then200 μL of TBS was added to the precipitate, and suspended to obtain anultracentrifugation precipitate fraction of K562 cell culturesupernatant. Protein concentration of the ultracentrifugationprecipitate fraction of K562 cell culture supernatant was measured usinga Protein Assay BCA Kit (produced by Wako Pure Chemical Industries,Ltd.), according to a protocol attached to the kit. The dilution seriesof the ultracentrifugation precipitate fraction of the calciumion-containing K562 cell culture supernatant was prepared by dilutionwith TBS containing 2 mM CaCl₂, so as to attain a protein concentrationof each 125, 250, 500, 1000, and 2000 ng/mL.

<(3) Preparation of HRP-Labeled Anti-CD63 Mouse Monoclonal Antibody>

The anti-CD63 mouse monoclonal antibody H5C6 (100 μL, 50 μg) (producedby BD Biosciences) was labeled with HRP using a Peroxidase LabelingKit-NH₂ (manufactured by Dojindo Molecular Technologies, Inc.),according to a protocol attached to the kit to obtain 200 μL of theHRP-labeled anti-CD63 mouse monoclonal antibody. Glycerol (200 μL) wasadded to and mixed with the resulting labeled antibody, and stored at−20° C. as the HRP-labeled anti-CD63 mouse monoclonal antibody stocksolution. The HRP-labeled anti-CD63 mouse monoclonal antibody stocksolution was diluted 2,000-fold with the 2 mM CaCl₂-containing TBS toprepare a diluted solution of the HRP-labeled anti-CD63 mouse monoclonalantibody.

<(4) Preparation of Immobilizing Solutions of PS Protein and Anti-MarkerProtein Antibody>

Immobilizing solutions of various PS proteins and the anti-markerprotein antibodies were prepared as follows. The anti-CD9 mousemonoclonal antibody M-L 13 (16 μL, 8 μg) (produced by BD Biosciences)and 784 μL of a 50 mM MOPS (pH 7.5) solution were mixed to prepare 800μL of the anti-CD9 antibody immobilizing solution. The anti-CD 63 mousemonoclonal antibody H5C6 (16 μL, 8 μg) (produced by BD Biosciences) and784 μL of the 50 mM MOPS (pH 7.5) solution were mixed to prepare 800 μLof the anti-CD 63 antibody immobilizing solution. The anti-CD81 mousemonoclonal antibody JS-81 (16 μL, 8 μg) (produced by BD Biosciences) and784 μL of the 50 mM MOPS (pH 7.5) solution were mixed to prepare 800 μLof the anti-CD81 antibody immobilizing solution. The His tag fusion typemouse MFG-E8 protein (80 μL, 8 μg) (produced by R & D Systems Inc.) and720 μL of the 50 mM MOPS (pH 7.5) solution were mixed to prepare 800 μLof the mMFG-E8 immobilizing solution. The His tag fusion type humanMFG-E8 protein (80 μL, 8 μg) (produced by R & D Systems Inc.) and 720 μLof the 50 mM MOPS (pH 7.5) solution were mixed to prepare 800 μL of thehMFG-E8 immobilizing solution. The His tag fusion type human Annexin Vprotein (40 μL, 8 μg) (produced by Creative BioMart) and 760 μL of the50 mM MOPS (pH 7.5) solution were mixed to prepare 800 μL of thehAnnexin V immobilizing solution. The Fc tag fusion type mouse Tim-1protein (32 μL, 8 μg) (produced by Wako Pure Chemical Industries, Ltd.)and 768 μL of the 50 mM MOPS (pH 7.5) solution were mixed to prepare 800μL of the mTim-1 immobilizing solution. The His tag fusion type mouseTim-2 protein (80 μL, 8 μg) (produced by R & D Systems Inc.) and 720 μLof the 50 mM MOPS (pH 7.5) solution were mixed to prepare 800 μL of themTim-2 immobilizing solution. The Fc tag fusion type mouse Tim-3 protein(32 μL, 8 μg) (produced by Wako Pure Chemical Industries, Ltd.) and 768μL of the 50 mM MOPS (pH 7.5) solution were mixed to prepare 800 μL ofthe mTim-3 immobilizing solution. The Fc tag fusion type mTim-4 protein(32 μL, 8 μg) (produced by Wako Pure Chemical Industries, Ltd.) and 768μL of the 50 mM MOPS (pH 7.5) solution were mixed to prepare 800 μL ofthe mTim-4 immobilizing solution. The Fc tag fusion type human Tim-1protein (32 μL, 8 μg) (produced by Wako Pure Chemical Industries, Ltd.)and 768 μL of the 50 mM MOPS (pH 7.5) solution were mixed to prepare 800μL of the hTim-1 immobilizing solution. The Fc tag fusion type humanTim-3 protein (32 μL, 8 μg) (produced by Wako Pure Chemical Industries,Ltd.) and 768 μL of the 50 mM MOPS (pH 7.5) solution were mixed toprepare 800 μL of the hTim-3 immobilizing solution. The Fc tag fusiontype human Tim-4 protein (32 μL, 8 μg) (produced by Wako Pure ChemicalIndustries, Ltd.) and 768 μL of the 50 mM MOPS (pH 7.5) solution weremixed to prepare 800 μL of the hTim-4 immobilizing solution.

<(5) ELISA Measurement>

The PS protein immobilizing solution or the anti-marker protein antibodyimmobilizing solution was dispensed into six wells, by 100 μL per wellfor one type of a protein, to an Immuno-plate Maxisorp C96 (manufacturedby Nunc A/S), and allowed to stand at 8° C. overnight. After removingthe dispensed solution, a blocking solution (TBS containing 25% BlockAce) was added to each well in each amount of 300 μL, and subjected to areaction at room temperature for 1 hour, while mixing at 500 rpm using aplate mixer. After the blocking solution was removed, for the wells onwhich the resulting PS protein or anti-marker protein antibody wasimmobilized, each 100 μL of the dilution series of the calciumion-containing K562 cell culture supernatant ultracentrifugationprecipitate fraction, having each a protein concentration of 125, 250,500, 1000, or 2000 ng/mL, was added to each of 5 wells, and 100 μL ofTBS containing 2 mM CaCl₂ was each added, as a blank, to 1 well, and themixture was subjected to a reaction at room temperature for 1 hour,while mixing at 500 rpm using the plate mixer. After washing three timeswith 300 μL of TBS-T containing 2 mM CaCl₂, 100 μL of the dilutedHRP-labeled anti-CD63 mouse monoclonal antibody solution was added toeach well, and the mixture was subjected to a reaction at roomtemperature for 1 hour, while mixing at 500 rpm using the plate mixer.After washing five times with 300 μL of TBS-T containing 2 mM CaCl₂, 100μL of the TMB solution (produced by Wako Pure Chemical Industries, Ltd.)was added to each well, and subjected to a standing reaction at roomtemperature for 30 minutes. After stopping the reaction by the additionof 100 μL of 1 M HCl, absorbance at 450 nm was measured with amicroplate reader Tecan Ultra (produced by Tecan Ltd.).

It should be noted that the proteins immobilized on the plate andpossibility of detection (results), in each Example and ComparativeExample, are shown in the following Table 32.

TABLE 32 Comparative Comparative Comparative Comparative ComparativeComparative Comparative Example 26 Example 27 Example 28 Example 29Example 30 Example 31 Example 32 Protein immobilized — Anti-CD9Anti-CD63 Anti-CD81 His tag fusion type His tag fusion type His tagfusion type on a plate mouse- human- human- derived MFG-E8 derivedMFG-E8 derived hAnnexin V Capability of X Δ Δ Δ Δ Δ Δ detection Name oflanes Blank Anti-CD9 Anti-CD63 Anti-CD81 mMFG-E8 hMFG-E8 hAnnexin V inFIG. 19 Example Comparative 100 Example 33 Example 101 Example 102Example 103 Example 104 Example 105 Protein immobilized Fc tag fusion Fctag fusion Fc tag fusion Fc tag fusion Fc tag fusion Fc tag fusion Fctag fusion on a plate type mouse- type mouse- type mouse- type mouse-type human- type human- type human- derived Tim-1 derived Tim-2 derivedTim-3 derived Tim-4 derived Tim-1 derived Tim-3 derived Tim-4 proteinprotein protein protein protein protein protein Capability of ◯ Δ ◯ ⊚ ◯◯ ⊚ detection Name of lanes mTim-1 mTim-2 mTim-3 mTim-4 hTim-1 hTim-3hTim-4 in FIG. 19<Results>

The results of the ELISA measurement obtained are shown in FIG. 19. FIG.19 shows each of detection signals (absorbance at 450 nm) derived fromthe HRP-labeled anti-CD63 mouse monoclonal antibody bound to exosomescontained in each protein concentration dilution series of the calciumion-containing K562 cell culture supernatant ultracentrifugationprecipitate fraction in the wells on which the PS protein or theanti-marker protein antibody was immobilized. The horizontal axis showsthe type of the PS protein or the anti-marker protein antibodyimmobilized on the well, and in the detection results for the wells onwhich the same PS protein or the like was immobilized, proteinconcentration in the calcium ion-containing K562 cell culturesupernatant ultracentrifugation precipitate fraction used is 0, 125,250, 500, 1000, 2000 ng/mL in the order from the left. In addition, thevertical axis shows the detection signal derived from the HRP-labeledanti-CD63 mouse monoclonal antibody bound to the exosome contained ineach protein concentration dilution series of the K562 cell culturesupernatant ultracentrifugation precipitate fraction.

In the wells, on which each of the Tim-1 protein, the Tim-3 protein, orthe Tim-4 protein was immobilized, the extracellular membrane vesiclesare detectable in higher sensitivity (Examples 100 to 105), as comparedwith the wells on which the anti-CD9 antibody, the anti-CD63 antibody orthe anti-CD81 antibody, which is the anti-marker protein antibody, waseach immobilized, the wells on which the MFG-E8 or the Annexin V, whichis a PS protein, was each immobilized, and the well on which the Tim-2protein, which is the Tim family protein, was immobilized (ComparativeExamples 27 to 33). That is, it has been revealed that the detectionmethod of the present invention, using the Tim protein pertaining to thepresent invention, is capable of detecting the extracellular membranevesicles in higher sensitivity, as compared with the conventionalmethods. In particular, it has been revealed that the extracellularmembrane vesicles can be detected in particularly high sensitivity byusing the Tim-4 protein among the Tim proteins.

Examples 106 to 109. Comparison of Sensitivity in Detection ofExtracellular Membrane Vesicles by ELISA by Difference in Tim ProteinImmobilization Method

As described below, detection sensitivity of the extracellular membranevesicles was compared in the case where the Tim-1 protein or the Tim-4protein was each immobilized directly on a plate, with the case wherethe SH-group biotin-labeled Tim-1 protein or the SH-group biotin-labeledTim-4 protein was each immobilized on the plate via streptavidin.

<(1) Preparation of Ultracentrifugation Precipitate Fractions of K562Cell Culture Supernatant>

The dilution series of the ultracentrifugation precipitate fractions ofthe calcium ion-containing K562 cell culture supernatant was prepared bythe same method as in “(2) Preparation of ultracentrifugationprecipitate fraction of calcium ion-containing K562 cell culturesupernatant” of Examples 100 to 105 and Comparative Examples 26 to 33,except for “diluting the ultracentrifugation precipitate fraction of thecalcium ion-containing K562 cell culture supernatant with TBS containing2 mM CaCl₂, so as to attain a protein concentration of each 500, 1000ng/mL”, instead of “diluting the ultracentrifugation precipitatefraction of the calcium ion-containing K562 cell culture supernatantwith TBS containing 2 mM CaCl₂, so as to attain a protein concentrationof each 125, 250, 500, 1000, 2000 ng/m L”.

<(2) Preparation of HRP-Labeled Anti-CD63 Mouse Monoclonal Antibody>

The diluted solution of the HRP-labeled anti-CD63 mouse monoclonalantibody was prepared by the same method as in “(3) Preparation ofHRP-labeled anti-CD63 mouse monoclonal antibody” of Examples 100 to 105and Comparative Examples 26 to 33.

<(3) Preparation of Biotin-Labeling and Immobilizing Solution of Tim-1Protein and Tim-4 Protein>

The Fc tag fusion type mouse-derived Tim-1 protein 40 μL, (10 μg)(produced by Wako Pure Chemical Industries, Ltd.), and 40 μL (10 μg) ofthe Fc tag fusion type mouse-derived Tim-4 protein (produced by WakoPure Chemical Industries Ltd.) were each labeled with biotin using theBiotin Labeling Kit-SH (produced by Dojindo Molecular Technologies,Inc.), according to a protocol attached to the kit to obtain theSH-group biotin-labeled Fc tag fusion type mTim-1 protein, and theSH-group biotin-labeled Fc tag fusion type mTim-4. Proteinconcentrations of the SH-group biotin-labeled Fc tag fusion type mTim-1protein, and the SH-group biotin-labeled Fc tag fusion type mTim-4protein were each measured using the Protein Assay BCA Kit (produced byWako Pure Chemical Industries, Ltd.), according to a protocol attachedto the kit. The SH-group biotin-labeled Fc tag fusion type mTim-1protein solution and the SH-group biotin-labeled Fc tag fusion typemTim-4 protein solution were each prepared by diluting these with TBS,so as to attain a protein concentration of 5 μg/mL.

The Fc tag fusion type mouse-derived Tim-1 protein (produced by WakoPure Chemical Industries, Ltd.), or the Fc tag fusion type mouse-derivedTim-4 protein (8 μL, (2 μg) (produced by Wako Pure Chemical IndustriesLtd.), and 392 μL of the 50 mM MOPS (pH 7.5) solution were mixed toprepare each of 400 μL of the mTim-1 protein immobilizing solution orthe mTim-4 protein immobilizing solution.

In addition, 2 μL (10 μg) of streptavidin (produced by Wako PureChemical Industries, Ltd.), and 998 μL of the 50 mM MOPS (pH 7.5)solution were mixed to prepare 1000 μL of the streptavidin immobilizingsolution.

<(4) Immobilization>

Each of the protein immobilizing solutions was added to three wells byeach 100 μL (0.5 μg) per well of the Immuno-plate Maxisorp C96(manufactured by Nunc A/S), and the plate was allowed to stand at 8° C.overnight. After removal of the added solution, a blocking solution (TBScontaining 25% Block Ace) was added to each well each 300 μL, and themixture was subjected to a reaction at room temperature for 1 hour,while mixing at 500 rpm using the plate mixer.

In addition, the streptavidin immobilizing solution was added to sixwells by each 100 μL (0.5 μg) per well of the Immuno-plate Maxisorp C96(produced by Nunc A/S), and the plate was allowed to stand at 8° C.overnight. After removal of the added solution, the blocking solution(TBS containing 25% Block Ace) was added to each well each 300 μL, andthe mixture was subjected to a reaction at room temperature for 1 hourwhile mixing at 500 rpm using the plate mixer. After removal of theblocking solution, the SH-group biotin-labeled Fc tag fusion type mTim-1protein solution and the SH-group biotin-labeled Fc tag fusion typemTim-4 protein solution were added to 3 wells by each 100 μL (0.5 μg)per well, and the mixture was subjected to a reaction at roomtemperature for 1 hour, while mixing at 500 rpm using the plate mixer.

<(5) ELISA Measurement>

The ELISA measurement was carried out by the same method as in Examples100 to 105 and Comparable Examples 26 to 33, except for each adding“each 100 μL of the calcium ion-containing dilution series of theultracentrifugation precipitate fraction of the K562 cell culturesupernatant, having a protein concentration of 500 or 1000 ng/mL, toeach well of two wells on which each protein was immobilized,” insteadof each adding “for the wells on which the resulting PS protein oranti-marker protein antibody was immobilized, each 100 μL of thedilution series of the calcium ion-containing K562 cell culturesupernatant ultracentrifugation precipitate fraction, having each aprotein concentration of 125, 250, 500, 1000, or 2000 ng/mL, was addedto each of 5 wells”.

It should be noted that the proteins immobilized on the plate, themethods for immobilizing the protein on the plate, and possibility ofdetection (result), in each Example and Comparative Example, are shownin the following Table 33. Possibility of detection in Table 33 is eachcomparison between Examples 106 and 107, and between Examples 108 and109.

TABLE 33 Example 106 Example 107 Example 108 Example 109 Immobilized Fctag fusion type SH-group biotin-labeled Fc tag fusion type SH-groupbiotin-labeled protein mouse-derived Tim-1 Fc tag fusion typemouse-derived Tim-4 Fc tag fusion type protein mouse-derived Tim-1protein mouse-derived Tim-4 protein protein Immobilization method Directimmobilization Biotin-streptavidin Direct immobilizationBiotin-streptavidin binding binding Possibility of detection ◯ ⊚ ◯ ⊚Name of lane mTim-1 bmTim-1/SA mTim-4 bmTim-4/SA in FIG. 20

The results of the ELISA measurement obtained are shown in FIG. 20. FIG.20 shows detection signals (absorbance at 450 nm) derived from theHRP-labeled anti-CD63 mouse monoclonal antibody bound to exosomescontained in each protein concentration dilution series of theultracentrifugation precipitate fraction of the calcium ion-containingK562 cell culture supernatant, in the wells on which the mTim-1 proteinwas directly immobilized on a plate, the wells on which the mTim-4protein was directly immobilized on a plate, the wells on which themTim-1 protein was immobilized by biotin-streptavidin binding, and thewells on which the mTim-4 protein was immobilized by biotin-streptavidinbinding.

The horizontal axis shows the type of the Tim proteins immobilized onthe well. In addition, the vertical axis shows detection signal derivedfrom the HRP-labeled anti-CD63 mouse monoclonal antibody bound to theexosome contained in each protein concentration dilution series of theultracentrifugation precipitate fraction of the calcium ion-containingK562 cell culture supernatant.

From FIG. 20, it has been revealed that the extracellular membranevesicles in the sample can be detected, according to ELISA using theTim-1 protein or the Tim-4 protein, irrespective of the immobilizationmethod of the Tim protein on the plate (Examples 106 to 109). Inparticular, the extracellular membrane vesicles can be detected inhigher sensitivity in the wells on which the Tim protein was immobilizedvia streptavidin (Example 107, wherein the plate and the Tim proteinwere bound by binding of biotin of the SH-group of the mTim-1 andstreptavidin, and Example 109, wherein the plate and the Tim proteinwere bound by binding of biotin of the SH-group of the mTim-4 andstreptavidin), as compared with the wells in which the Tim protein wasimmobilized directly on the plate (Example 106, wherein the mTim-1 wasimmobilized directly to the plate, and Example 108, wherein the mTim-4was immobilized directly on the plate).

Therefore, it has been revealed that, as the binding form between theTim protein and a solid phase, such a case is more preferable where theplate and the Tim protein are bound by binding of biotin of the SH-groupof the Tim protein and streptavidin.

Examples 110 to 115. Comparative Example 34 to 39. Comparison ofSensitivity in Detection of Viruses by ELISA Using PS Protein

As described below, baculovirus in the baculovirus-containing insectcell culture supernatant sample was detected by carrying out a sandwichELISA using a plate on which various PS proteins or the antibody againstbaculovirus surface marker protein gp64 was immobilized, and using theHRP-labeled anti-gp64 antibody as a detection antibody.

<(1) Preparation of Co-Transfection Cell Culture Supernatant>

Co-transfection cell culture supernatant was prepared by the same methodas in “(1) Preparation of co-transfection cell culture supernatant” ofExamples 84 to 95 and Comparative Examples 20 to 21.

<(2) Preparation of Calcium Ion-Containing and Baculovirus-ContainingInsect Cell Culture Supernatant Sample>

The calcium ion-containing baculovirus solution was obtained similarlyas in “(2) Preparation of baculovirus-containing insect cell culturesupernatant sample” of Examples 84 to 95 and Comparative Examples 20 to21.

<(3) Preparation of Dilution Series of Baculovirus-Containing InsectCell Culture Supernatant Sample>

The baculovirus-containing insect cell culture supernatant sample (50μL) and 4950 μL of the PSFM-J1 medium were mixed to prepare a 100-folddiluted solution, then 2.0 mL of the 100-fold diluted solution and 2.0mL of the PSFM-J1 medium were mixed to prepare 4.0 mL of the 200-folddiluted solution, then 2.0 mL of the 200-fold diluted solution and 2.0mL of the PSFM-J1 medium were mixed to prepare 4.0 mL of a 400-folddiluted solution, and then 2.0 mL of the 400-fold diluted solution and2.0 mL of the PSFM-J1 medium were mixed to prepare 4.0 mL of a 800-folddiluted solution.

<(4) Preparation of HRP-Labeled Anti-Gp64 Mouse Monoclonal Antibody>

The anti-gp64 mouse monoclonal antibody AcV1 200 μL, (100 μg) (producedby Novus Biologacals LLC) was labeled with HRP using the PeroxidaseLabeling Kit-NH₂ (produced by Dojindo Molecular Technologies, Inc.),according to a protocol attached to the kit to obtain 200 μL of theHRP-labeled anti-gp64 mouse monoclonal antibody solution. TheHRP-labeled anti-gp64 mouse monoclonal antibody solution was diluted1,000-fold with TBS containing 2 mM CaCl₂ to prepare a diluted solutionof the HRP-labeled anti-gp64 mouse monoclonal antibody.

<(5) Preparation of Immobilizing Solutions of PS Protein and Anti-Gp64Antibody>

As described below, the immobilizing solutions of various PS proteinsand marker proteins were prepared. The anti-gp64 mouse monoclonalantibody AcV1 (12 μL, 6 μg) (produced by Novus Biologicals LLC) and 588μL of the 50 mM MOPS (pH 7.5) solution were mixed to prepare 600 μL ofthe anti-gp64 antibody immobilizing solution. The His tag fusion typemouse-derived MFG-E8 protein (60 μL, 6 μg) (produced by R & D SystemsInc.) and 540 μL of the 50 mM MOPS (pH 7.5) solution were mixed toprepare 600 μL of the mMFG-E8 immobilizing solution. The His tag fusiontype human-derived MFG-E8 protein (60 μL, 6 μg) (produced by R & DSystems Inc.) and 540 μL of the 50 mM MOPS (pH 7.5) solution were mixedto prepare 600 μL of the hMFG-E8 immobilizing solution. The His tagfusion type human-derived Annexin V protein (30 μL, 6 μg) (produced byCreative BioMart) and 570 μL of the 50 mM MOPS (pH 7.5) solution weremixed to prepare 600 μL of the hAnnexin V immobilizing solution. The Fctag fusion type mouse-derived Tim-1 protein (24 μL, 6 μg) (produced byWako Pure Chemical Industries, Ltd.) and 576 μL of the 50 mM MOPS (pH7.5) solution were mixed to prepare 600 μL of the mTim-1 immobilizingsolution. The His tag fusion type mouse-derived Tim-2 protein (60 μL, 6μg) (produced by R & D Systems Inc.) and 540 μL of the 50 mM MOPS (pH7.5) solution were mixed to prepare 600 μL of the mTim-2 immobilizingsolution. The Fc tag fusion type mouse-derived Tim-3 protein (24 μL, 6μg) (produced by Wako Pure Chemical Industries, Ltd.) and 576 μL of the50 mM MOPS (pH 7.5) solution were mixed to prepare 600 μL of the mTim-3immobilizing solution. The Fc tag fusion type mouse-derived Tim-4protein (24 μL, 6 μg) (produced by Wako Pure Chemical Industries, Ltd.)and 576 μL of the 50 mM MOPS (pH 7.5) solution were mixed to prepare 600μL of mTim-4 immobilizing solution. The Fc tag fusion type human-derivedTim-1 protein (24 μL, 6 μg) (produced by Wako Pure Chemical Industries,Ltd.) and 576 μL of the 50 mM MOPS (pH 7.5) solution were mixed toprepare 600 μL of the hTim-1 immobilizing solution. The Fc tag fusiontype human-derived Tim-3 protein (24 μL, 6 μg) (produced by Wako PureChemical Industries, Ltd.) and 576 μL of the 50 mM MOPS (pH 7.5)solution were mixed to prepare 600 μL of the hTim-3 immobilizingsolution. The Fc tag fusion type human-derived Tim-4 protein (24 μL, 6μg) (produced by Wako Pure Chemical Industries, Ltd.) and 576 μL of the50 mM MOPS (pH 7.5) solution were mixed to prepare 600 μL of the hTim-4immobilizing solution.

<(6) ELISA Measurement>

The PS protein immobilizing solution or anti-gp64 antibody immobilizingsolution was dispensed into the Immuno-plate Maxisorp C96 (produced byNunc A/S), each five wells for one type of a protein, by 100 μL perwell, and allowed to stand at 8° C. overnight. After removing thedispensed solution, each 300 μL of a blocking solution (TBS containing25% Block Ace) was added to each well, and subjected to a reaction atroom temperature for 1 hour, while mixing at 500 rpm using the platemixer. After removal of the blocking solution, for the resulting wellson which the PS protein or the anti-gp64 antibody was immobilized, each100 μL of the calcium ion-containing and baculovirus-containing insectcell culture supernatant sample dilution series (100-fold diluted,200-fold diluted, 400-fold diluted, 800-fold diluted) prepared in (3)was added to each well of 4 wells, and 100 μL of the PMSF-J1 medium as ablank was added to 1 well, and the mixture was subjected to a reactionat room temperature for 1 hour, while mixing at 500 rpm using the platemixer. After washing three times with 300 μL of the 2 mMCaCl₂-containing TBS-T, 100 μL of the HRP-labeled anti-gp64 mousemonoclonal antibody diluted solution was added to each well, and themixture was subjected to a reaction at room temperature for 1 hour,while mixing at 500 rpm using the plate mixer. After washing five timeswith 300 μL of the 2 mM CaCl₂-containing TBS-T, 100 μL of the TMBsolution (produced by Wako Pure Chemical Industries, Ltd.) was added toeach well, and subjected to a standing reaction at room temperature for30 minutes. After stopping the reaction by the addition of 100 μL of 1 MHCl, absorbance at 450 nm was measured with the microplate reader TecanUltra (produced by Tecan Ltd.).

It should be noted that proteins immobilized on the plate, andpossibility of detection (results), in each Example and ComparativeExample, are shown in the following Table 34.

TABLE 34 Comparative Comparative Comparative Comparative ComparativeExample 34 Example 35 Example 36 Example 37 Example 38 Example 110Protein immobilized — Anti-gp64 antibody His tag fusion His tag fusionHis tag fusion Fc tag fusion on a plate type mouse- type human- typehuman- type mouse- derived MFG-E8 derived MFG-E8 derived Annexin Vderived Tim-1 protein Capability of X Δ Δ Δ Δ ◯ detection Name of lanesBlank Anti-gp64 mMFG-E8 hMFG-E8 hAnnexin V mTim-1 in FIG. 21 ComparativeExample 39 Example 111 Example 112 Example 113 Example 114 Example 115Protein immobilized His tag fusion Fc tag fusion Fc tag fusion Fc tagfusion Fc tag fusion Fc tag fusion on a plate type mouse- type mouse-type mouse- type human- type human- type human- derived Tim-2 derivedTim-3 derived Tim-4 derived Tim-1 derived Tim-3 derived Tim-4 proteinprotein protein protein protein protein Capability of Δ ◯ ⊚ ◯ ◯ ⊚detection Name of lanes mTim-2 mTim-3 mTim-4 hTim-1 hTim-3 hTim-4 inFIG. 21<Results>

The results of the ELISA measurement obtained are shown in FIG. 21. FIG.21 shows detection signal (absorbance at 450 nm) derived from theHRP-labeled anti-gp64 mouse monoclonal antibody bound to exosomescontained in each dilution series of the calcium ion-containing andbaculovirus-containing insect cell culture supernatant in the wells, onwhich various PS proteins or the anti-gp64 antibody was immobilized. Thehorizontal axis shows the types of the PS proteins or the anti-markerprotein antibody immobilized on the well, and the detection results forthe wells, on which the same PS protein or the like was immobilized,were shown in the order from left, blank, 800-fold dilution, 400-folddilution, 200-fold dilution, and 100-fold dilution, in the dilutionseries of the calcium ion-containing and baculovirus-containing insectcells culture supernatant sample used. In addition, the vertical axisshows detection signal (absorbance at 450 nm) derived from theHRP-labeled anti-gp64 mouse monoclonal antibody.

In the wells, on which the Tim-1 protein, the Tim-3 protein, or theTim-4 protein was each immobilized, viruses were detectable in highersensitivity (Examples 110 to 115), as compared with the wells, on whichthe anti-gp64 antibody which is an antibody against a surface markerprotein of baculovirus was immobilized, the wells on which the PSprotein MFG-E8, or the Annexin V was immobilized was immobilized, andthe well, on which the Tim family Tim-2 protein was immobilized(Comparative Example 34 to 39). That is, it has been revealed that thedetection method of the present invention using the Tim proteinpertaining to the present invention is capable of detecting viruses inhigher sensitivity, as compared with the conventional methods. Inparticular, it has been revealed that the viruses can be detected inparticularly higher sensitivity by using the Tim-4 protein among the Timproteins.

Examples 116 to 119. Obtaining of Viruses by Carrier on which TimProtein is Immobilized

Obtaining of the viruses pertaining to the present invention was carriedout using the carrier on which the biotin-labeled Tim-4 protein wasimmobilized.

<(1) Preparation of Co-Transfection Cell Culture Supernatant>

The co-transfection cell culture supernatant was prepared similarly asin “(1) Preparation of co-transfection cell culture supernatant” ofExamples 84 to 95 and Comparative Examples 20 to 21.

<(2) Preparation of Calcium Ion-Containing and Baculovirus-ContainingInsect Cell Culture Supernatant Sample>

The calcium ion-containing and baculovirus-containing insect cellculture supernatant sample was obtained by carrying out similarly as in“(2) Preparation of baculovirus-containing insect cell culturesupernatant sample” of Examples 84 to 95 and Comparative Examples 20 to21.

<(3) Preparation of Diluted Solution of Baculovirus-Containing InsectCell Culture Supernatant Sample>

The calcium ion-containing and baculovirus-containing insect cellculture supernatant sample (100 μL) was mixed with 800 μL of the PSFM-J1medium to prepare 900 μL of a nine-fold diluted solution.

<(4) Biotin Labeling of SH-Group of Tim-4 Protein>

For 100 μL of the PBS solution containing the Fc tag fusion typemouse-derived Tim-4 protein (produced by Wako Pure Chemical Industries,Ltd.) (containing 10 μg of the Fc tag fusion type mouse-derived Tim-4protein), the SH-group of the Fc tag fusion type mouse-derived Tim-4protein was labeled with biotin using the Biotin Labeling Kit-SH(produced by Dojindo Molecular Technologies, Inc.), according to aprotocol attached to the kit to obtain 100 μL of the PBS solutioncontaining the SH-group biotin-labeled Fc tag fusion type mTim-4protein.

<(5) Dilution of Tag Fused Tim Protein>

The PBS solution (5 μL) containing the Fc tag fusion type mTim-4 protein(produced by Wako Pure Chemical Industries, Ltd.) (containing 0.5 μg ofthe Fc tag fusion type mouse-derived Tim-4 protein) was mixed with 95 μLof TBS to obtain 100 μL of a solution containing 0.5 μg of thebiotin-unlabeled Fc tag fusion type mTim-4 protein.

The PBS solution (5 μL) containing the SH-group biotin-labeled Fc tagfusion type mTim-4 protein prepared in the (4) (containing 0.5 μg of theSH-group biotin-labeled Fc tag fusion type mTim-4 protein) was mixedwith 95 μL of TBS to obtain 100 μL of a solution containing 1 μg of thebiotin-labeled Fc tag fusion type mTim-4 protein.

<(6) Washing of Beads>

The PBS-T solution containing 30 μg/μL Dynabeads Protein G (5 μL)(produced by Thermo Fisher Scientific Inc.) (containing 0.3 mg of theDynabeads Protein G) was transferred to a 1.5 mL tube (manufactured byBM Equipment Co., Ltd.), and after washing twice with 500 μL of TBS-T(TBS, 0.0005% Tween 20), the tube was loaded on a magnetic stand, andthe washing solution was removed.

In addition, 30 μL of the PBS solution containing a MagCapture Tamavidin2-REV (produced by Wako Pure Chemical Industries, Ltd.) (containing 0.3mg of the beads) was transferred to the 1.5 mL tube (manufactured by BMEquipment Co., Ltd.), and after washing twice with 500 μL of TBS-T (TBS,0.0005% Tween 20), the tube was loaded on a magnetic stand to remove thewashing solution.

<(7) Immobilization of Tim Protein on Beads>

A solution (100 μL) containing 0.5 μg of the SH-group biotin-unlabeledFc tag fusion type mTim-4 protein was added to the 1.5 mL tubecontaining 0.3 mg of the Dynabeads Protein G, in a pellet state, afterthe washing operation, and subjected to a reaction at 8° C. for 1 hour,under 1200 rpm using a thermomixer to obtain the carrier on which theSH-group biotin-unlabeled Fc tag fusion type mTim-4 was bound (it may beabbreviated as “the biotin-unlabeled Fc tag fusion type mTim-4 carrier”,in some cases).

In addition, 100 μL of a solution containing 0.5 μg of the SH-groupbiotin-labeled Fc tag fusion type mTim-4 protein was added to the 1.5 mLtube containing 0.3 mg of the MagCapture Tamavidin 2-REV after thewashing operation, and allowed to bind on the beads similarly as aboveto obtain the carrier on which the SH-group biotin-labeled Fc tag fusiontype mTim-4 was bound (it may be abbreviated as “the biotin-labeled Fctag fusion type mTim-4 carrier”, in some cases).

It should be noted that, the resulting these two types of carriers maybe abbreviated collectively as “the Tim protein-bound carrier” in somecases.

<(8) Obtaining of Baculovirus by Obtaining Method of the PresentInvention>

The resulting these Tim protein-bound carriers above were subjected tothe washing operation three times with 250 μL of TBS-T (TBS, 0.0005%Tween 20) to obtain the Tim protein-bound carrier in a pellet state.Subsequently, 100 μL of the diluted solution of the calciumion-containing and baculovirus-containing insect cell culturesupernatant prepared in the (3) was added to each of the resulting twokinds of Tim protein-bound carriers, in pellet state, and the mixturewas subjected to a reaction at 8° C., for 12 hours under 1200 rpm usinga thermomixer. Thereafter, the Tim protein-bound carrier after thereaction was subjected to the washing operation three times with each250 μL of the calcium ion-containing TBS-T (Tris buffer, 0.0005% Tween20, 2 mM CaCl₂). At the third time of the washing operation, 250 μL ofthe CaCl₂-containing TBS-T solution containing the Tim protein-boundcarrier was each dispensed into two 1.5 mL tubes by 125 μL, and thetubes were loaded on a magnetic stand and then the washing solution wasremoved.

The 1% SDS aqueous solution (50 μL) or 25 μL of the TBS solutioncontaining 1 mM EDTA, each as the eluent, was added to 0.15 mg of twokinds of the Tim protein-bound carriers, in a pellet state, and thenthey were mixed using a vortex mixer for 10 seconds and spun down. TheTBS solution containing 1 mM EDTA (25 μL) was added again to the tube towhich EDTA had been added, and the same operation was carried out toobtain the eluate.

It should be noted that the types of Tim proteins, carriers, and the Timprotein-(non) bound carriers, used in each Example, are shown in thefollowing Table 35.

TABLE 35 Example 116 117 118 119 Tim protein-bound Carrier DynabeadsDynabeads MagCapture MagCapture carrier protein G protein G TamavidinREV-2 Tamavidin REV-2 Tim protein Biotin-unlabeled Biotin-unlabeledSH-group SH-group mouse-derived mouse-derived biotin-labeled Fc tagbiotin-labeled Fc tag Tim-4 protein Tim-4 protein fusion type fusiontype mouse-derived mouse-derived Tim-4 protein Tim-4 protein<(9) Western Blotting>

Western blotting was carried out by the same method as in “(5) Westernblotting” of Examples 80 to 83 and Comparative Examples 16 to 19, exceptfor “adding 15 μL of the 4×sample buffer (produced by Wako Pure ChemicalIndustries, Ltd.) to each 15 μL of the eluate obtained in the (8)“Obtaining of baculovirus by obtaining method of the present invention”,instead of “adding 3.75 μL of the 4× sample buffer (produced by WakoPure Chemical Industries, Ltd.) to each 11.25 μL of the resultingeluate”, and using “the anti-baculovirus gp64 antibody” instead of “theanti-Lamp-1 antibody”.

It should be noted that the types of Tim proteins, carriers, and theeluents used for obtaining viruses from the Tim protein-bound carriers,used in each Example, and the lane numbers in Western blotting are shownin the following Table 36.

TABLE 36 Example 116 117 118 119 Tim protein-bound Carrier DynabeadsDynabeads MagCapture MagCapture carrier protein G protein G TamavidinREV-2 Tamavidin REV-2 Tim protein Biotin-unlabeled Biotin-unlabeledSH-group SH-group mouse-derived mouse-derived biotin-labeled Fc tagbiotin-labeled Fc tag Tim-4 protein Tim-4 protein fusion type fusiontype mouse-derived mouse-derived Tim-4 protein Tim-4 protein Eluent 1% 1mM 1% 1 mM SDS EDTA SDS EDTA Lane numbers in FIG. 22 1 2 3 4<Results>

The results of Western blotting obtained are shown in FIG. 22. In FIG.22, each lane is the result of the following:

Lane 1: Results of Example 116 (the results of the case of using thecarrier in which the SH-group biotin-unlabeled Fc tag fusion type mTim-4protein was bound on the Dynabeads protein G, and using the 1% SDSaqueous solution as the eluent);

Lane 2: Results of Example 117 (the results of the case of using thecarrier in which the SH-group biotin-unlabeled Fc tag fusion type mTim-4protein was bound on the Dynabeads protein G, and using the TBS solutioncontaining 1 mM EDTA as the eluent);

Lane 3: Results of Example 118 (the results of the case of using thecarrier in which the SH-group biotin-labeled Fc tag fusion type mTim-4protein was bound on the MagCapture Tamavidin REV-2, and using the 1%SDS aqueous solution as the eluent);

Lane 4: Results of Example 119 (the results of the case of using thecarrier in which the SH-group biotin-labeled Fc tag fusion type mTim-4protein was bound on the MagCapture Tamavidin REV-2, and using the TBSsolution containing 1 mM EDTA as the eluent).

From FIG. 22, it has been revealed that the viruses cannot be obtainedby using the carrier on which the SH-group biotin-unlabeled or labeledTim-4 protein was bound, from the fact that a band of gp64, which is amarker protein of baculovirus, was observed in any cases of the lanes 1to 4.

In addition, it has been revealed that more viruses can be obtained inimmobilization on beads (carrier) via the SH-group of the Tim proteinthan immobilization via the tag, in the case where the elution iscarried out with the calcium chelating agent, from comparison betweenlane 2 and lane 4

Examples 120 to 121, Comparative Examples 40 to 41. Comparison ofDetection Sensitivity of Extracellular Membrane Vesicles by FlowCytometer

As described below, the extracellular membrane vesicles derived from theK562 cell culture supernatant was detected by the flow cytometer, usingthe carrier on which the Tim-4 protein or the antibody against CD63 of asurface marker protein of exosome was immobilized, and the PE-labeledanti-CD63 antibody as the detection antibody.

<(1) Preparation of Calcium Ion-Containing Culture Supernatant (StockSolution)>

The human chronic myelogenous leukemia cell strain K562, 1×10⁷ cells,secreting the extracellular membrane vesicles, was cultured for 3 daysunder condition at 37° C. and 5% CO₂, using 80 mL of the X-VIVO 15medium (produced by Lonza AG). Then the cells were precipitated bycentrifugal separation treatment (300×G, for 5 minutes) to remove thesupernatant. The precipitated cells were suspended in 60 mL of theX-VIVO 15 medium containing 10 μM monensin sodium (produced by MPBiomedicals Co., Ltd) and cultured for 24 hours under condition at 37°C. and 5% CO₂.

Thereafter, the culture solution was subjected to centrifugal separationtreatment (300×G, for 5 minutes) to recover the culture supernatant. Therecovered culture supernatant (60 mL) was further subjected tocentrifugal separation treatment three times (the first time: 300×G, for3 minutes, the second time: 1,200×G, for 20 minutes, the third time:10,000×G, for 20 minutes) to separate impurities and obtain asupernatant (hereinafter it may be abbreviated as “the culturesupernatant (stock solution)”, in some cases).

In addition, CaCl₂ was added to the resulting culture supernatant stocksolution, so as to attain the final concentration of 2 mM, to obtain the2 mM CaCl₂-containing K562 cell culture supernatant concentratedsolution sample (hereinafter it may be abbreviated as “the calciumion-containing culture supernatant (stock solution)”, in some cases).

<(2) Preparation of Diluted Solution of Culture Supernatant Sample>

The resulting calcium ion-containing culture supernatant (stocksolution) (500 μL), and 500 μL of the X-VIVO 15 medium were mixed toprepare 1.0 mL of a 2-fold diluted solution, then 200 μL of the 2-folddiluted solution and 800 μL of the X-VIVO 15 medium were mixed toprepare 1.0 mL of a 10-fold diluted solution to obtain each of the2-fold diluted calcium ion-containing culture supernatant solution(stock solution) and the 10-fold diluted calcium ion-containing culturesupernatant solution (stock solution).

<(3) Biotin Labeling of SH-Group of Tim-4 Protein>

For 100 μL of the PBS solution containing the Fc tag fusion typemouse-derived Tim-4 protein (produced by Wako Pure Chemical Industries,Ltd.) (containing 10 μg of the Fc tag fusion type mouse-derived Tim-4protein), the SH-group of the Fc tag fusion type mouse-derived Tim-4protein was labeled with biotin, using the Biotin Labeling Kit-SH(produced by Dojindo Molecular Technologies, Inc.), according to aprotocol attached to the kit to obtain 100 μL of the PBS solutioncontaining the SH-group biotin-labeled Fc tag fusion type mouse-derivedTim-4 protein.

<(5) Washing of Beads>

An Exosome-Streptavidin Isolation/Detection Reagent (20 μL) (containingthe Dynabeads Streptavidin 4.5 μm, 1×10⁷/mL) (produced by Thermo FisherScientific Inc.) was dispensed into each of two 1.5 mL tubes(manufactured by BM Equipment Co., Ltd.), and the washing operation wascarried out using 200 μL of TBS.

An Exosome-Human CD63 Isolation/Detection Reagent (20 μL) (containingthe Dynabeads Anti-CD63 antibody immobilized, 4.5 μm, 1×10⁷/mL)(produced by Thermo Fisher Scientific Inc.) was dispensed into each oftwo 1.5 mL tubes (manufactured by BM Equipment Co., Ltd.), and thewashing operation was carried out using 200 μL of TBS.

<(6) Immobilization of Tim-4 Protein on Beads>

A solution (100 μL) containing 0.1 μg of the SH-group biotin-labeled Fctag fusion type mTim-4 protein was added to two 1.5 mL tubes containingthe Dynabeads Streptavidin, in a pellet state, after the washingoperation, and subjected to a reaction at 8° C. for 30 minutes under1200 rpm using a thermomixer to obtain a solution containing the carrieron which the SH-group biotin-labeled Fc tag fusion type mTim-4 (it maybe abbreviated as “the biotin-labeled Fc tag fusion type mTim-4carrier”, in some cases) was bound, (the solution may be abbreviated as“the solution containing biotin-labeled Fc tag fusion type mTim-4carrier”, in some cases).

<(7) Reaction with Extracellular Membrane Vesicles>

The resulting solution (100 μL) containing the biotin-labeled Fc tagfusion type mTim-4 carrier above was loaded on a magnetic stand, andthen the solution in the solution containing the biotin-labeled Fc tagfusion type mTim-4 carrier was removed. The biotin-labeled Fc tag fusiontype mTim-4 carrier, in a pellet state, and the Exosome-Human CD63Isolation/Detection Reagent, in a pellet state, after the washingoperation, were washed twice with 200 μL of TBS containing 0.1% BSA toobtain carriers, in a pellet state. Subsequently, 100 μL of the 2-folddiluted solution of the calcium ion-containing culture supernatant(stock solution), and the 10-fold diluted solution of the culturesupernatant (stock solution) prepared in the (2) were each added to eachof the resulting pellet-like carriers and subjected to a reaction at 8°C. for 12 hours under 1,200 rpm using a thermomixer. After that, thecarrier after the reaction was subjected to the washing operation oncewith each 300 μL of TBS containing the calcium ion-the 0.1% BSA (TBS,0.1% BSA, 2 mM CaCl₂), and once with 400 μL of TBS containing thecalcium ion-0.1% BSA, and each carrier, in a pellet state, after thewashing operation, was suspended in 300 μL of TBS containing the calciumion-the 0.1% BSA (TBS, 0.1% BSA, 2 mM CaCl₂) to obtain two types ofsuspension of the carriers described in the following Table 37.

TABLE 37 1 2 Carrier Dynabeads Exosome-Human CD63 StreptavidinIsolation/Detection Reagent Tim SH-group biotin-labeled Fc — protein tagfusion type mouse-derived Tim-4 protein<(8) Detection by PE-Labeled Anti-CD63 Antibody>

The resulting suspension of two types of the carriers in the (7) waseach dispensed into two 1.5 mL tubes in each amount of 100 μL, andpelletized by magnetic separation, and then each 20 μL of the PE-labeledanti-CD63 mouse monoclonal antibody H5C6 (produced by BD Biosciences),or the PE-labeled control mouse IgG (produced by BD Biosciences) wasadded and subjected to a reaction 25° C. for 1 hour under 1000 rpm usinga thermomixer. Thereafter, the washing operation was carried out twicewith each 300 μL of TBS containing the calcium ion, and the 0.1% BSA(TBS, 0.1% BSA, 2 mM CaCl₂), and then the carriers were suspended in 500μL of TBS containing the calcium ion, and the 0.1% BSA (TBS, 0.1% BSA, 2mM CaCl₂). Thereafter, the filtrate filtered through a Falcon CellStrainer (produced by Corning Inc.) was recovered, and measurement andanalysis were carried out with a flow cytometer Gallios (manufactured byBeckman Coulter, Inc.). It should be noted that laser wavelength at thetime of measurement was 488 nm, and detection wavelength was 575 nm.

It should be noted that the types of Tim proteins, carriers and thesamples, used in each Example, are shown in the following Table 38.

TABLE 38 Example 120 Example 121 Comparative Example 40 ComparativeExample 41 Carrier Dynabeads Dynabeads Exosome-Human CD63 Exosome-HumanCD63 Streptavidin Streptavidin Isolation/Detection ReagentIsolation/Detection Reagent Tim SH-group biotin-labeled Fc SH-groupbiotin-labeled Fc — — protein tag fusion type tag fusion typemouse-derived Tim-4 mouse-derived Tim-4 protein protein Sample 2-folddiluted 10-fold diluted 2-fold diluted 10-fold diluted calciumion-containing calcium ion-containing calcium ion-containing calciumion-containing culture supernatant culture supernatant culturesupernatant culture supernatant (stock solution) (stock solution) (stocksolution) (stock solution)<Results>

The results of the flow cytometer measurement are shown in FIG. 23. FIG.23 shows ratio of a signal detected exosomes captured using thebiotin-labeled Fc tag fusion type mTim-4 carrier and the anti-CD63antibody carrier from the calcium ion-containing culture supernatant(stock solution), diluted at each magnification by the PE-labeledanti-CD63 mouse monoclonal antibody, and a noise detected the same bythe PE-labeled control mouse IgG (Signal/Noise ratio). The horizontalaxis shows dilution magnification of the calcium ion-containing culturesupernatant (stock solution) used, and the vertical axis shows ratio ofthe signal detected by the PE-labeled anti-CD63 mouse monoclonalantibody and the noise detected by the PE-labeled control mouse IgG.

From FIG. 23, it has been revealed that the extracellular membranevesicles can be detected by using the carrier on which the Tim proteinpertaining to the present invention is bound. In addition, it has beenshown that the flow cytometer measurement can be carried out in higherSignal/Noise ratio (detection sensitivity) by using the carrier on whichthe Tim-4 is immobilized, as compared with using the carrier on whichthe anti-CD63 antibody is immobilized. That is, it has been revealedthat the extracellular membrane vesicles can be detected in highersensitivity by performing the flow cytometric analysis using the Timcarrier pertaining to the present invention, as compared with theconventional method.

Examples 122 to 123, Comparative Example 42 to 43. Obtaining of Virusesby Carrier on which Tim Protein is Immobilized

Obtaining of the viruses pertaining to the present invention was carriedout using the carrier on which the Tim protein was immobilized.

<(1) Biotin Labeling of SH-Group in Fc Tag Fusion Type mTim-4 Protein>

The SH-group of the Fc tag fusion type mouse-derived Tim-4 protein in100 μL of the PBS solution containing the Fc tag fusion typemouse-derived Tim-4 protein (produced by Wako Pure Chemical Industries,Ltd.) (containing 10 μg of the Fc tag fusion type mouse-derived Tim-4protein) was labeled with biotin using the Biotin Labeling Kit-SH(produced by Dojindo Molecular Technologies, Inc.), according to aprotocol attached to the kit to obtain 100 μL of the PBS solutioncontaining the SH-group biotin-labeled Fc tag fusion type mTim-4protein.

<(2) Dilution of SH-Group Biotin-Labeled Fc Tag Fusion Type mTim-4Protein>

The SH-group biotin-labeled Fc tag fusion type mTim-4 protein-containingTBS solution (10 μL) prepared in the (1) (containing 1 μg of theSH-group biotin-labeled Fc tag fusion type mTim-4 protein) was mixedwith 190 μL of TBS to obtain 200 μL of a solution containing 1 μg of theSH-group biotin-labeled Fc tag fusion type mTim-4 protein.

<(3) Washing of Beads>

The PBS solution (60 μL) containing the MagCapture Tamavidin 2-REV(produced by Wako Pure Chemical Industries, Ltd.) (containing 0.6 mg ofthe MagCapture Tamavidin 2-REV) was transferred to two 1.5 mL tubes(manufactured by BM Equipment Co., Ltd.), and after washing each twicewith 500 μL of TBS-T (TBS, 0.0005% Tween 20), the tube was loaded on amagnetic stand to remove the washing solution.

<(4) Immobilization of SH-Group Biotin-Labeled Fc Tag Fusion Type mTim-4Protein on Beads>

The solution (200 μL) containing 1 μg of the SH-group biotin-labeled Fctag fusion type mTim-4 protein, or 200 μL of TBS was added to the two1.5 mL tubes containing 0.6 mg of the MagCapture Tamavidin 2-REV, afterthe washing operation, and each was subjected to a reaction at 8° C. for1 hour at 1200 rpm using a thermomixer to obtain the carrier on whichthe SH-group biotin-labeled Fc tag fusion type mTim-4 was bound (it maybe abbreviated as “the biotin-labeled Fc tag fusion type mTim-4-boundcarrier”, in some cases), and the carrier on which SH-groupbiotin-unlabeled Fc tag fusion type mTim-4 was bound (it may beabbreviated as “the biotin-labeled Fc tag fusion type mTim-4-non-boundcarrier”, in some cases). It should be noted that, the Tim protein-boundcarrier and the Tim protein-non-bound carrier may be abbreviatedcollectively as “the Tim protein-(non-)bound carrier” in some cases.

<(5) Obtaining of Influenza Virus by Obtaining Method of the PresentInvention>

The resulting 2 types of the Tim protein-(non-)bound carriers above weresubjected to the washing operation three times with each 500 μL of TBS-T(Tris buffer, 0.0005% Tween 20). An influenza A virus solution (60 μL)(H1N1 Å/WS/33 strain) (ATCC Code: VR-825), containing the calcium ionadded with calcium chloride at the final concentration of 2 mM, wasadded to each of the two types of the Tim protein-(non-)bound carriersafter the washing operation, and subjected to a reaction at 8° C. for 2hours at 1200 rpm using a thermomixer. The two types of the Timprotein-(non-bound-)carriers after the reaction were subjected to thewashing operation three times with each 500 μL of the calciumion-containing TBS-T (Tris buffer, 0.0005% Tween 20, 2 mM CaCl₂). Afterthe third time of the washing operation, 500 μL of the calciumion-containing TBS-T (Tris buffer, 0.0005% Tween 20, 2 mM CaCl₂) wasadded to each of the two types of the Tim protein-(non-)bound carriersand suspended to obtain each 500 μL of suspension. The suspension wasdispensed by 250 μL into each of the two 1.5 mL tubes, and after thetubes were loaded on a magnetic stand, the washing solution was removedto obtain each Tim protein-(non-)bound carrier in a pellet state.

It should be noted that the types of the carriers and the Tim protein,used in each Example and Comparative Example, are shown in the followingTable 39.

TABLE 39 Comparative Comparative Example 42 Example 43 Example 122Example 123 Carrier MagCapture MagCapture MagCapture MagCaptureTamavidin2- Tamavidin2- Tamavidin2- Tamavidin2- REV REV REV REV Tim — —Biotin-labeled Biotin-labeled protein Fc tag fusion Fc tag fusion typeTim-4 type Tim-4 protein protein

Among the four 1.5 ml tubes containing the resulting 0.3 mg of the Timprotein-(non-)bound carrier in a pellet state, for one 1.5 mL tubecontaining the Tim protein-bound carrier, and one 1.5 mL tube containingthe Tim protein-non-bound carrier, each 20 μL of the 1% SDS aqueoussolution was added as the eluent, then they were mixed using a vortexmixer at room temperature for 10 seconds, and spun down to obtain eacheluate.

In addition, among the four 1.5 ml tubes containing 0.3 mg of the Timprotein-(non-)bound carrier in the pellet state, for one 1.5 mL tubecontaining the Tim protein-bound carrier and one 1.5 mL tube containingthe Tim protein-non-bound carrier, each 10 μL of the TBS solutioncontaining 50 mM EDTA as the eluent was added, and they were mixed usinga vortex mixer at room temperature for 10 seconds, and spun down. TheTBS solution containing 50 mM EDTA was again added to each of the tubesafter spun down, and they were mixed using a vortex mixer at roomtemperature for 10 seconds, and spun down to obtain each eluate.

The TBS solution (2.2 μL) containing 10% β-propiolactone was added toeach 20 μL of the resulting four types of the eluates, and they weremixed using a vortex mixer and left to stand on ice for 3 hours toobtain four types of 22.2 μL of β-propiolacton-treated eluates.

<(6) Western Blotting>

The TBS solution (3.3 μL) containing 10% β-propiolactone was added to 30μL of the calcium ion-containing influenza A virus solution (H1N1A/WS/33 strain) (ATCC Code: VR-825) and they were mixed using a vortexmixer. Then, 11.1 μL of the 4×sample buffer (produced by Wako PureChemical Industries, Ltd.) was added to the solution which had been leftto stand on ice for 3 hours, and incubated at 95° C. for 3 minutes toobtain an input sample for Western blotting (44.4 μL in total). Inaddition, 7.4 μL of the 4× sample buffer (produced by Wako Pure ChemicalIndustries, Ltd.) was added to each 22.2 μL of the 4 types of theeluates treated with β-propiolactone, and incubated at 95° C. for 3minutes to obtain each of the eluate samples for Western blotting (29.6μL in total). The input sample (20 μL) for Western blotting and each 20μL of the eluate sample for Western blotting were loaded on the SuperSep Ace 5-20% gel (produced by Wako Pure Chemical Industries, Ltd.), andsubjected to electrophoresis at 30 mA for 60 minutes. The resultingelectrophoresis gel was transcribed onto the PVDF membrane (produced byMillipore Corporation) at 1.2 mA/cm² for 60 minutes using the semi-dryblotter and the discontinuous buffer (Anode buffer 1: 0.3 M Tris/20%methanol, Anode buffer 2: 0.025 M Tris/20% methanol, Cathode buffer:0.025 M Tris/0.04 M aminocaproic acid/20% methanol). The 5% skimmedmilk, diluted with TBS-T (TBS buffer, 0.1% Tween 20), was added to thepost-transcription PVDF membrane, and subjected to a reaction at roomtemperature for 1 hour for blocking, and then subjected to a reaction atroom temperature for 1 hour, with 2 mL of the anti-influenza A virusNucleoprotein antibody C43 (produced by Abcam plc), diluted 300-foldwith TBS-T. The PVDF membrane after the reaction was washed three timeswith TBS-T, and reacted with the secondary antibody {anti-mouse IgG(H+L), donkey, IgG fraction, peroxidase-conjugated antibody} (producedby Jackson Immuno Research Laboratories, Inc.), diluted 30,000-fold withTBS-T at room temperature for 1 hour. After washing 5 times with TBS-T,the ImmunoStar Zeta (produced by Wako Pure Chemical Industries, Ltd.)was added to the PVDF membrane after being subjected to a reaction witheach antibody, and a luminescent signal was detected using the LAS-4000(manufactured by GE Healthcare).

It should be noted that the types of carriers, Tim protein, and theeluents for elution from the carrier, used in each Example, are shown inthe following Table 40.

TABLE 40 Comparative Comparative Example 42 Example 43 Example 122Example 123 Carrier MagCapture MagCapture MagCapture MagCaptureTamavidin2- Tamavidin2- Tamavidin2- Tamavidin2- REV REV REV REV Tim — —Biotin-labeled Biotin-labeled protein Fc tag fusion Fc tag fusion typeTim-4 type Tim-4 protein protein Eluent 1% 1 mM 1% 1 mM SDS EDTA SDSEDTA Lane 2 3 4 5 numbers in FIG. 24<Results>

The results of Western blotting obtained are shown in FIG. 24. In FIG.24, each lane is the result of the following.

Lane 1: Results of electrophoresis of influenza A virus;

Lane 2: Results of Comparative Example 42 (the results of the case ofusing the biotin-labeled Fc tag fusion type mTim-4-non-bound carrier,and using the 1% SDS solution as the eluent);

Lane 3: Results of Comparative Example 43 (the results of the case ofusing the biotin-labeled Fc tag fusion type mTim-4-non-bound carrier,and using the TBS solution containing 50 mM EDTA as the eluent);

Lane 4: Results of Example 122 (the results of the case of using thebiotin-labeled Fc tag fusion type mTim-4-bound carrier, and using the 1%SDS solution as the eluent);

Lane 5: Results of Example 123 (the results of the case of using thebiotin-labeled Fc tag fusion type mTim-4-bound carrier, and using theTBS solution containing 50 mM EDTA as the eluent).

From FIG. 24, it has been revealed that the influenza virus can beobtained by using the carrier on which the Tim protein was bound, fromthe fact that no band of the Nucleoprotein, which is a marker protein ofinfluenza A virus, was observed in any of the cases in lanes 2 to 3 (inthe case where the biotin-labeled Fc tag fusion type mTim-4-non-boundcarrier was used), whereas the band of the Nucleoprotein, which is amarker protein of influenza A virus, was observed in any of the cases inthe lanes 4 to 5 (in the case where the biotin-labeled Fc tag fusiontype mTim-4-bound carrier was used).

In addition, it has been revealed that the influenza virus can beobtained efficiently by using the carrier on which the Tim protein isbound, from comparison between the lane 1 (the result of electrophoresisof the influenza A virus) and the lanes 4 to 5 (results of using thebiotin-labeled Fc tag fusion type mTim-4-bound carrier).

Examples 124 to 125, Comparative Examples 44 to 45. Obtaining of Virusesby Carrier on which Tim Protein is Immobilized

Obtaining of the viruses pertaining to the present invention was carriedout using the carrier on which the Tim protein was immobilized.

<(1) Preparation of RS Virus-Containing HEp-2 Cell Culture SupernatantSample>

A 10% FBS-containing D-MEM high glucose medium (20 mL) (produced by WakoPure Chemical Industries, Ltd.) containing 1.5×10⁶ cells of the HEp-2cells was added to a 75 cm² flask and cultured for 4 days undercondition at 37° C. and 5% CO₂. The supernatant was removed from theculture solution after the culture, and 5 mL of the E-MEM mediumcontaining 2% FBS and 5.3×10⁵ TCID₅₀/mL RS virus (ATCC, Code: VR-26) wasadded, and allowed to stand for 1 hour under condition at 36° C. and 5%CO₂. Additional 20 mL of the E-MEM medium containing 2% FBS was addedand cultured for 3 days under condition at 36° C. and 5% CO₂. Thesupernatant was removed from the culture solution after the culture, and20 mL of the E-MEM medium containing 2% FBS was added to culture thecells for 2 days under condition at 36° C. and 5% CO₂. Thereafter, thecells were released from the flask by pipetting, recovered together withthe culture supernatant into a 50 mL centrifuge tube, and centrifuged at1,500 rpm for 10 minutes to recover only the culture supernatant.Calcium chloride was added to the resulting culture supernatant, so asto attain the final concentration of 2 mM, and used it as “thecalcium-containing RS virus solution”.

<(2) Biotin Labeling of SH-Group in Fc Tag Fusion Type mTim-4 Protein>

The PBS solution (100 μL) containing the SH-group biotin-labeled Fc tagfusion type mTim-4 protein was obtained by carrying out the same methodas in <(1) Biotin labeling of SH-group in Fc tag fusion type mTim-4protein> of Examples 122 to 123 and Comparative Example 42 to 43.

<(3) Dilution of SH-Group Biotin-Labeled Fc Tag Fusion Type mTim-4Protein>

The solution (200 μL) containing 1 μg of the SH-group biotin-labeled Fctag fusion type mTim-4 protein was obtained by carrying out the samemethod as in <(2) Dilution of SH-group biotin-labeled Fc tag fusion typemTim-4 protein> of Examples 122 to 123 and Comparative Examples 42 to43.

<(4) Washing of Beads>

The MagCapture Tamavidin 2-REV, after carrying out the washingoperation, was obtained by carrying out the same method as in <(3)Washing of beads> of Examples 122 to 123 and Comparative Examples 42 to43.

<(5) Immobilization of SH-Group Biotin-Labeled Fc Tag Fusion Type mTim-4Protein on Beads>

The biotin-labeled Fc tag fusion type mTim-4 protein-bound carrier, andthe biotin-labeled Fc tag fusion type mTim-4 protein-(non)-bound carrierwere each obtained by carrying out the same method as in <(4)Immobilization of SH-group biotin-labeled Fc tag fusion type mTim-4protein on beads> of Examples 122 to 123 and Comparative Examples 42 to43.

It should be noted that the biotin-labeled Fc tag fusion type mTim-4protein-bound carrier and the biotin-labeled Fc tag fusion type mTim-4protein-non-bound carrier may be abbreviated collectively as “the Timprotein-non-bound carrier”, in some cases.

<(6) Obtaining of RS Virus by Obtaining Method of the Present Invention>

Each 22.2 μL of the β-propiolacton-treated eluate was obtained bycarrying out the same method as in <(5) Obtaining of influenza virus byobtaining method of the present invention> of Examples 122 to 123 andComparative Examples 42 to 43, except for using “120 μL of the calciumion-containing RS virus solution prepared in the (1)”, instead of “60 μLof the influenza A virus solution (H1N1 A/WS/33 strain) (ATCC Code:VR-825) containing the calcium ion added with calcium chloride at thefinal concentration of 2 mM”.

It should be noted that the types of carriers and the Tim protein, usedin each Example, are shown in the following Table 41.

TABLE 41 Comparative Comparative Example 44 Example 45 Example 124Example 125 Carrier MagCapture MagCapture MagCapture MagCaptureTamavidin2- Tamavidin2- Tamavidin2- Tamavidin2- REV REV REV REV Tim — —Biotin-labeled Biotin-labeled protein Fc tag fusion Fc tag fusion typeTim-4 type Tim-4 protein protein<(7) Western Blotting>

Western blotting was carried out by the same method as in <(6) Westernblotting> of Examples 122 to 123 and Comparative Examples 42 to 43,except for using “30 μL of the calcium ion-containing RS virus solution”instead of “30 μL of the calcium ion-containing influenza A virussolution (H1N1 A/WS/33 strain) (ATCC Code: VR-825)”, and using “2 mL ofthe anti-RS virus F protein antibody 2F7 (produced by Abcam plc),diluted 500-fold with TBS-T”, instead of “2 mL of the anti-influenza Avirus nucleoprotein antibody C43 (produced by Abcam plc), diluted300-fold with TBS-T”, and a luminescent signal was detected

It should be noted that the types of the carriers, the Tim protein, andthe eluents from the carriers, used in each Example, are shown in thefollowing Table 42.

TABLE 42 Comparative Comparative Example 44 Example 45 Example 124Example 125 Carrier MagCapture MagCapture MagCapture MagCaptureTamavidin2- Tamavidin2- Tamavidin2- Tamavidin2- REV REV REV REV Tim — —Biotin-labeled Biotin-labeled protein Fc tag fusion Fc tag fusion typeTim-4 type Tim-4 protein protein Eluent 1% 1 mM 1% 1 mM SDS EDTA SDSEDTA Lane 2 3 4 5 numbers in FIG. 25<Results>

The results of Western blotting obtained are shown in FIG. 25. In FIG.25, each lane is the result of the following.

Lane 1: Results of electrophoresis of the RS virus;

Lane 2: Results of Comparative Example 44 (the results of the case ofusing the biotin-labeled Fc tag fusion type mTim-4-non-bound carrier,and using the 1% SDS solution as the eluent);

Lane 3: Results of Comparative Example 45 (the results of the case ofusing the biotin-labeled Fc tag fusion type mTim-4-non-bound carrier,and using the TBS solution containing 50 mM EDTA as the eluent);

Lane 4: Results of Example 124 (the results of the case of using thebiotin-labeled Fc tag fusion type mTim-4-bound carrier, and using the 1%SDS solution as the eluent);

Lane 5: Results of Example 125 (the results of the case of using thebiotin-labeled Fc tag fusion type mTim-4-bound carrier, and using theTBS solution containing 50 mM EDTA as the eluent).

From in FIG. 25, it has been revealed that the RS virus can be obtainedby using the carrier on which the Tim protein is bound, from the factthat no band of the F protein, which is a marker protein of the RSvirus, was observed in any of the cases in the lanes 2 to 3 (in the casewhere the biotin-labeled Fc tag fusion type mTim-4-non-bound carrier wasused), whereas a band of the F protein, which is a marker protein of RSvirus, was observed in any of the cases in the lanes 4 to 5 (in the casewhere the biotin-labeled Fc tag fusion type mTim-4-bound carrier wasused).

In addition, it has been revealed that the RS virus can be obtainedefficiently by using the carrier on which the Tim protein is bound, fromcomparison between the lane 1 (the result of electrophoresis of the RSvirus) and the lanes 4 to 5 (result of using the biotin-labeled Fc tagfusion type mTim-4-bound carrier).

INDUSTRIAL APPLICABILITY

The Tim protein-bound carrier of the present invention, an obtainingmethod and a removing method for the extracellular membrane vesicle andvirus using the carrier, and the kit comprising the carrier are capableof obtaining or removing the extracellular vesicle and virus present inthe sample in higher purity or in a more intact state, as well as moreeasily and efficiently, as compared with conventional methods forobtaining the extracellular membrane vesicle and virus, and the kitcomprising the antibody or the like to be used for the obtaining method.

The Tim protein-bound carrier of the present invention, and theobtaining method of the present invention are useful, since theextracellular membrane vesicle obtained by the obtaining method of thepresent invention are utilized as pharmaceuticals and the like, and theobtained virus are utilized as vaccines, or vectors, and the like.

The Tim protein-bound carrier of the present invention and the removingmethod of the present invention are the useful ones, since the samplesfrom which the extracellular membrane vesicle or virus is removed by theremoving method of the present invention are those prevented fromcontamination of biological extracellular membrane vesicle or envelopedvirus, and are utilized for research, or the like, using the sample.

The Tim protein-bound carrier of the present invention, the detectionmethod for the extracellular membrane vesicle and the virus using thecarrier are capable of detecting the extracellular membrane vesicle andthe virus in higher sensitivity, as compared with the conventionaldetecting methods for detecting the extracellular membrane vesicle andvirus.

The Tim protein-bound carrier of the present invention and the detectionmethod of the present invention are useful, because according to thedetection method of the present invention, even in the case of detectinga trace amount of the extracellular membrane vesicle and virus in thespecimen, it is possible to carry out the measurement as it is withoutconcentration or purification of the specimen.

The invention claimed is:
 1. A method for obtaining an extracellularmembrane vesicle in the sample, comprising the following steps: (1) astep for forming a complex of Tim-4 protein bound to a carrier, and theextracellular membrane vesicle in a sample, in the presence of a calciumion (complex formation step), (2) a step for separating the complex andthe sample (complex separation step), and (3) a step for separating theextracellular membrane vesicle from the complex using a calcium ionchelating agent to obtain the extracellular membrane vesicle (obtainingstep).
 2. The method according to claim 1, wherein the complex formationstep is a step for forming the complex of Tim-4 protein bound to thecarrier, and the extracellular membrane vesicle in the sample, bycontacting Tim-4 protein bound to the carrier and the extracellularmembrane vesicle in the sample, in the presence of the calcium ion. 3.The method according to claim 1, wherein the complex formation step is astep for forming the complex of Tim-4 protein bound to the carrier, andthe extracellular membrane vesicle in the sample, by contacting Tim-4protein, the carrier and the extracellular membrane vesicle in thesample, in the presence of the calcium ion.
 4. The method according toclaim 1, wherein Tim-4 protein comprises an IgV domain.
 5. The methodaccording to claim 2, wherein Tim-4 protein comprises an IgV domain. 6.The method according to claim 3, wherein Tim-4 protein comprises an IgVdomain.
 7. The method according to claim 1, wherein the carrier binds toTim-4 protein via a SH-group of the Tim-4 protein.
 8. The methodaccording to claim 2, wherein the carrier binds to Tim-4 protein via aSH-group of the Tim-4 protein.
 9. The method according to claim 3,wherein the carrier binds to Tim-4 protein via a SH-group of the Tim-4protein.